Molecular Vision 2004; 10:112-121 <http://www.molvis.org/molvis/v10/a15/> Received 17 November 2003 | Accepted 19 February 2004 | Published 19 February 2003

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Expression of nonphagocytic NADPH oxidase system in the ocular lens

Ponugoti Vasantha Rao,^1,2 Rupalatha Maddala,¹ Faith John,³ Jacob Samuel Zigler, Jr.³
 
 

Departments of ¹Ophthalmology and ²Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC; ³National Eye Institute, NIH, Bethesda, MD

Correspondence to: P. Vasantha Rao, Ph.D., Department of Ophthalmology, Box 3802, Duke University Medical Center, Durham, NC, 27710; Phone: (919) 681-5883; FAX: (919) 684-8983; email: rao00011@mc.duke.edu

Abstract

Purpose: The primary goal of this study was to characterize the Rac GTPase associated, NADPH oxidase-mediated Reactive Oxygen Species (ROS)-generating system in the lens tissue.

Methods: NADPH oxidase activity in lens tissue was determined by quantifying superoxide-induced lucigenin photoemission. Immunological and PCR/RT-PCR techniques were utilized to determine expression of different components of the NADPH oxidase system in lens tissue. Growth factor stimulated ROS production was determined quantitatively in human lens epithelial cells using dichlorofluorescein diacetate.

Results: Lens homogenates from different species showed generation of superoxide in a lucigenin-enhanced chemiluminescence assay in the presence of NADPH. This activity was found to be lens protein concentration dependent, heat sensitive, and inhibitable by superoxide dismutase and the flavoprotein inhibitor, diphenyleneiodonium (DPI). The distribution of superoxide generating activity in lens was confined predominantly to the lens epithelium, with very low levels in cortex and none in the nucleus. Immunological assays have demonstrated the presence of p67phox and p47phox in lens tissue, while PCR and RT-PCR reactions amplified DNA products corresponding to the p67phox, p40phox, p22phox, gp91phox, and Rac1 components of the NADPH oxidase complex from human and mouse lens cDNA libraries. Serum starved human lens epithelial cells stimulated with different growth factors including EGF, b-FGF, PDGF, TGF-β, and LPA demonstrated increased production of ROS, a response which was blocked by inhibitors of NADPH oxidase, such as DPI and the antioxidant-N-acetyl cysteine (NAC). RT-PCR analysis of human lens RNA confirmed readily detectable levels of expression of low molecular weight protein tyrosine phosphatase (LMW-PTP), which is a well-characterized target of redox signaling pathway(s).

Conclusions: These data demonstrate the presence of a functional nonphagocytic NADPH oxidase system in lens that is predominantly localized to the lens epithelium. Several growth factors appear to stimulate the activity of lens NADPH oxidase, resulting in increased production of ROS in lens epithelial cells, indicating that redox signaling may have an important role in growth factor effects on lens growth and development.

Introduction

Rac and Rho, members of the Rho family of small GTPases, play a critical role in the coupling of extracellular signals to reorganization of the actin cytoskeleton, formation of focal adhesions, and morphometric changes [1,2]. Further, there is extensive literature documenting the participation of these small GTPases in growth factor-mediated signaling pathways controlling various cellular processes including cytoskeletal reorganization, cell adhesion, gene expression, cell cycle progression, and cell survival [1-3]. In earlier studies, we demonstrated the expression of various small GTPases including Rho and Rac in lens tissue, and provided data to suggest a potential role for these small GTPases in maintaining lens growth and integrity [4-6]. Additionally, several different growth factors were found to stimulate both Rho and Rac activity and cause extensive cytoskeletal reorganization in a Rho- and Rac-dependent fashion in lens epithelial cells [7].

Rac GTPase, unlike Rho GTPase, plays an important role in NADPH oxidase-mediated production of superoxide in neutrophils [8,9]. Rac GTPase is a component of the membrane-assembled NADPH oxidase complex, and interacts directly with the oxidase flavocytochrome in addition to binding to the regulatory p67 subunit to regulate electron transfer from NADPH to molecular oxygen [9,10]. The NADPH oxidase is a major source of superoxide during phagocytosis and plays a vital role in nonspecific host defense [11-13]. It comprises a membrane-associated cytochrome b588, composed of one p22phox and one gp91phox subunit and at least four cytosolic subunits (p47phox, p67phox, p40phox, and the small GTPase rac1 or rac2) [11-13]. Rac GTPase has been shown to play an essential role in the activation of NADPH oxidase system [8,9].

Although the NADPH oxidase system was initially thought to be neutrophil-specific, it has become increasingly apparent in recent years that nonphagocytic cells such as fibroblasts, endothelial cells, epithelial cells, vascular smooth muscle cells, and other types of cells possess O_2^- producing enzymes analogous to the phagocytic NADPH oxidase [14-21]. The nonphagocytic NADPH oxidases are structurally related to, but functionally distinct from, the widely studied neutrophil oxidase. Under physiological conditions, nonphagocytic NADPH oxidases have very low-constitutive activity. However, enzyme activity can be upregulated both acutely and chronically in response to stimuli such as growth factors and cytokines [15]. ROS production by the oxidase has been demonstrated to serve a signaling role [22-27], and also implicated in oxidative damage [15,28,29]. In the last couple of years, there has been a huge increase in interest in redox-signaling in general, and ROS have been demonstrated to regulate various protein functions through reversible thiol-oxidation [23-27]. Therefore, in this study we were particularly interested in evaluating the existence of a functional Rac/NADPH oxidase system in lens tissue, because ROS generated by this system are thought to play an important role in redox signaling pathways which regulate cellular processes such as cell growth, apoptosis, extracellular matrix remodeling, and cytoskeletal reorganization [23-27]. Furthermore, ROS-mediated oxidative stress is also implicated as a pathogenic factor in the development of certain types of cataracts [30,31].

Towards this end, we evaluated the expression of different components of the NADPH oxidase system (at the transcript and protein level, respectively), and measured NADPH oxidase activity in lens tissues. Additionally, using selective inhibitors of NADPH oxidase and anti-oxidants, we confirmed the participation of the NADPH oxidase system in growth factor-induced ROS production in human lens epithelial cells.

Methods

Materials

Lens tissues from human, monkey, bovine, and rat were obtained from the following sources; NDRI, Philadelphia, PA; FDA, Bethesda, MD; a local slaughter house; and Taconic farms, respectively. Fresh mouse eyes were obtained from mice that were bred in the vivarium at Duke University Medical Center. Animals were utilized in accordance with the "ARVO Statement for the Use of Animals in Ophthalmic and Vision Research", under an approved animal protocol. Human lens cDNA libraries from adult and fetal lenses were provided by Pedro Gonzalez, Duke University Medical Center, Durham, NC and Toshi Shinohara, University Of Nebraska, Omaha, NB. Polyclonal antibodies raised against p67phox, p40 phox, and p47phox were kind gifts from Thomas Leto from the National Institute of Allergy and Infectious Diseases, NIH. Lucigenin, N-acetyl cysteine, superoxide dismutase, diphenyleneiodonium, 2-7-dichlorodihydrofluorescein diacetate (DCF-DA), and NADPH were procured from Sigma-Aldrich (St. Louis, MO). RNeasy mini kits and RT-PCR kits were purchased from Qiagen (Valencia, CA) and Clontech (Palo Alto, CA), respectively.

Tissue homogenates

Lens tissues from different species were homogenized in phosphate buffer (pH 7.0) containing 20 mM KH₂PO₄, 1 mM EGTA and 1 mM phenylmethanesulfonyl fluoride, using glass homogenizers. These homogenates were further subjected to sonication, and total tissue homogenates were utilized for measuring the activity of NADPH oxidase. Protein content of homogenates was determined by the Bradford method [32].

NADPH Oxidase assay

NADPH oxidase activity was determined based on superoxide induced lucigenin photoemission, as described by Cui and Douglas [33]. Enzyme assays were carried out in a final volume of 1 ml containing 50 mM phosphate buffer, pH 7.0, 1 mM EGTA, 150 mM sucrose, 0.5 mM lucigenin, 0.1 mM NADPH, and tissue homogenate. Enzyme reactions were initiated with the addition of lucigenin. Photoemission, expressed in terms of relative light units (RLU), was measured every min for 5 min using a luminometer. Assays were carried out in the dark at room temperature with appropriate controls.

ROS detection and quantification

Detection of intracellular ROS was carried out as described by Sundaresan et al. [25]. To detect the generation of intracellular ROS in lens epithelial cells, human lens epithelial cells (cell line SRA01/04 provided by V. N. Reddy, Kellogg Eye Center, Ann Arbor, MI) were grown in 6 well plastic dishes with DMEM media containing 10% fetal bovine serum and gentamicin (100 μg/ml). After cells reached 80% confluence, serum content was gradually dropped to 1% and cells were allowed to stay in this condition for 24 h. The medium was then replaced with minimal essential medium (MEM) lacking Phenol Red. Cells were maintained in MEM for 2 h at 37 °C in the incubator, followed by the addition of 2-7-dichlorofluorescin diacetate (DCF-DA; 5 μg/ml). Cells were then incubated at 37 °C for 10 min prior to the addition of different growth factors (20 ng/ml), either in the presence or absence of the anti-oxidant N-acetyl cysteine or the NADPH oxidase inhibitor-DPI. Immediately after the addition of growth factors, fluorescence images of cells were captured for 10 min, on a continuous basis, using a Zeiss phase contrast microscope (model IM 35) equipped with a fluorescent lamp source. Color images were captured in the dark, with a camera attached to the microscope. For those assays (performed in the presence of N-acetyl cysteine or DPI) these compounds were added to the cells one h prior to DCFDA addition. Fluorescence color images captured from the growth factor cells were compared with those obtained from untreated controls, and with cells pretreated with N-acetyl cysteine or DPI.

Quantitative measurement of intracellular ROS in human lens epithelial cells was performed by following the procedure of Suzukawa et al. [34]. Briefly, equal numbers of human lens epithelial cells (in the range of 1x10⁶) were seeded into 96 well culture plates and incubated for 48 h in DMEM medium containing 10% FBS and gentamicin (100 μg/ml) at 37 °C under 5% CO₂. Serum content in the medium was then dropped to 1%, and after 24 h, this medium was exchanged with MEM lacking Phenol-Red. Wherever anti-oxidant (N-acetyl cysteine) or DPI controls were required, cells were incubated with these compounds for one h prior to addition of DCFDA (5 μg/ml). Cells were incubated with DCFDA for 10 min, followed by addition of the respective growth factors (EGF, b-FGF, TGF-β, PDGF, and LPA) at a concentration of 20 ng/ml, except for LPA, which was used at 5 μg/ml. Following the addition of growth factors, cells were allowed to sit for 2 min, after which DCF fluorescence intensity was measured using a plate reader (Hewlett-Packard Cytofluor) at excitation/emission wavelengths of 485 and 530 nm, respectively. The relative units of DCF fluorescence obtained from different samples (untreated control, growth factor stimulated, and inhibitor treated) were compared for statistical significance using the Student t-test.

PCR/RT-PCR

For RT-PCR analysis, total RNA was extracted from human or mouse lenses or from human lens epithelial cells (SRA01/04) using a Qiagen RNeasy mini kit (Qiagen, Valencia, CA). DNase-1 treated samples of total RNA were reverse transcribed using an RT-PCR kit (Clontech, Palo Alto, CA). Expression of phox40, phox67, phox91, Rac1, and Rac2 in mouse and human lenses and expression of LMW-PTP in human lens or human lens epithelial cells was confirmed by RT-PCR using sequence-specific forward and reverse oligonucleotide primer pairs (Table 1).

Expression of p22 phox, p67phox, p91phox, and Rac1 in human lens was determined by polymerase chain reaction (PCR) using a cDNA library derived from adult human lens. Sequence specific forward and reverse oligonucleotide primer pairs were used to amplify the above-described NADPH oxidase components (Table 1).

Both, PCR and RT-PCR reactions were based on 30 cycle amplifications carried out employing a 30 s denaturation at 95 °C, 30 s annealing at 58 °C, and one min extension at 72 °C, in a GeneAmp PCR system 9700 (PE Applied Biosystems, Foster City, CA). PCR products were separated on 1.5% agarose gels and stained with ethidium bromide. Results were captured using Polaroid photography.

Western blot analysis

Aliquots of both soluble and insoluble (25,000x g pellet) fractions obtained from lenses of different species were subjected to SDS-polyacrylamide gel electrophoresis using 12.5% or 10% acrylamide gels. Resolved proteins were transferred to nitrocellulose membrane using a Bio-Rad transfer apparatus. Nitrocellulose membranes were blocked with 2% non-fat milk protein for 2 h, and then incubated overnight at 4 °C with the appropriate primary antibodies. After washing with Tris buffer containing 0.1% Tween 20, blots were incubated for 2 h at room temperature with peroxidase-conjugated secondary antibody. Finally, blots were developed using enhanced chemiluminescence reagents.

Results

NADPH oxidase activity in lens tissues

As an initial approach to evaluating the presence of NADPH-Oxidase activity in lens tissue, total homogenates prepared from fresh monkey lenses were tested for superoxide-induced lucigenin chemiluminescence using a luminometer. Monkey lens homogenate exhibited a decent dose-dependent linear increase of lucigenin chemiluminiscence, measured in relative light units (RLU)/mg of total lens protein (Figure 1A). The values of RLU obtained in the presence of lens homogenate were much higher than the values of samples lacking lens homogenate. To determine the distribution of lucigenin activating activity in the lens, monkey lenses were dissected into epithelium (capsule/epithelium), cortex, and nucleus. After peeling the lens capsule with fine forceps, the remaining lens tissue was divided into cortex and nucleus using a cork borer. Lens homogenates prepared from the whole lens or from distinct regions of the lens were utilized in the lucigenin assay. As can be seen from Figure 1B, most of the lucigenin chemiluminescence enhancing activity, expressed per mg of total protein, localized to the lens epithelium, with very little in the cortex and none in the nuclear region of the lens.

To confirm whether lucigenin chemiluminescence detected in the lens was superoxide-dependent, lucigenin assays were carried out in the presence of superoxide dismutase (SOD), at 50 units/ml. Inclusion of SOD caused a 65% reduction in the lucigenin activity (Figure 1C). Additionally, activation of lucigenin chemiluminescence was found to be heat sensitive, as heat inactivation of lens homogenates at 65 °C for 20 min abolished lucigenin photoemission (Figure 1C). Finally to address whether the activation of lucigenin chemiluminescence is catalyzed by NADPH oxidase, enzyme assays were carried out in the presence of diphenyleneiodonium (DPI), a flavoprotein inhibitor routinely used as an inhibitor of NADPH oxidase inhibitor [23,25]. Assays carried out in the presence of 50 μM of DPI revealed more than 80% inhibition compared to untreated controls, as shown in Figure 1C. Lenses from different species including rat, bovine, monkey, and human demonstrated detectable levels of NADPH oxidase activity (5,004; 818; 8,002 and 14,000 RLU/mg total protein, respectively; n=1).

Expression of NADPH Oxidase components in lens tissue

NADPH oxidase is a multicomplex enzyme consisting of cytosolic (p67phox, p47phox, p40phox, and Rac) and membrane bound (cytochrome b558, p91phox, and p22phox) subunits [12,13]. RT-PCR or PCR analysis was performed to confirm expression of different NADPH oxidase subunits from mouse lens RNA or from the human lens cDNA, respectively. Both mouse (Figure 2A) and human lenses (Figure 2B) revealed the expression of both cytochrome b558 subunits and cytosolic subunits of NADPH oxidase. Using sequence-specific forward and reverse oligonucleotide primers, DNA fragments of the expected size and corresponding to the different subunits of NADPH oxidase, were amplified. Figure 2 illustrates RT-PCR or PCR amplification of p40phox, p67phox, p91phox, rac1, and rac2 in mouse lens (Figure 2A) and p22phox, p67phox, p91phox, and Rac1 in human lens (Figure 2B). Interestingly, Rac2 expression was not found in human lens by PCR amplification but its expression was detected in day one mouse lens by RT-PCR analysis Figure 2A. Mouse lenses were also microdissected into epithelium, cortex, and nucleus and total RNA extracted from these regions were subjected to RT-PCR analysis to determine the distribution of various components of NADPH oxidase. Only lens epithelium showed detectable levels of DNA products for the individual components of NADPH oxidase; none were detected in the lens cortex or nucleus (data not shown).

We also attempted to confirm the presence of NADPH oxidase in lens tissue using an independent line of evidence. To this end, we screened both soluble (25,000x g supernatant) and insoluble homogenates obtained from lenses of different species, by western blot analysis, using specific polyclonal antibodies raised against recombinant p67phox, p47phox, p91phox, and p22phox. Human neutrophil homogenate was utilized as a positive control in these experiments. Both soluble and insoluble fractions from homogenates of rat, monkey, and human lenses exhibited positive immunoreactivity against p67phox (goat polyclonal antibody) and p47phox antibodies (goat polyclonal antibody), as depicted in Figure 3. While mouse lens soluble homogenates also showed positive immunoreactivity against both p67phox and p47phox antibodies (Figure 3), the insoluble fraction did not yield a clear immunopositive reaction against the p47phox antibody (data not shown). However, none of the lens homogenates exhibited immunopositivity for antibodies directed against the p91 phox and p22 phox subunits of cytochrome 588.

ROS production in human lens epithelial cells

To explore the significance of NADPH oxidase expression in lens, we have determined the effects of different growth factors on generation of ROS in human lens epithelial cells, since this enzyme is thought to be activated by external factors including growth factors [22,23]. Serum starved human lens epithelial cells were pretreated with DCF-DA for 10 min prior to being stimulated with different growth factors. Fluorescence images obtained from untreated and growth factor treated cells were compared for changes in the fluorescence intensity. Growth factor stimulation led to a transient enhancement of DCF-DA-dependent fluorescence, reaching a peak within two to three min and gradually decreasing to background levels by 10 min post-addition. A representative image of ROS-induced green fluorescence stimulated by 5 min of EGF exposure is shown in Figure 4. Similar effects, but of varying intensities, were observed with other growth factors including FGF, LPA, TGF-β, and PDGF (data not shown). The increased fluorescence induced by growth factors was found to be sensitive to N-acetyl cysteine and DPI, confirming that the enhanced fluorescence signal in lens cells is due to increased ROS production catalyzed by NADPH oxidase. Pretreatment of cells with either N-acetyl cysteine or DPI markedly reduced the growth factor-stimulated increase in green fluorescence, as shown in Figure 4.

To quantify the generation of ROS induced by growth factors in lens epithelial cells, human lens epithelial cells cultured in 96 well culture plates (n=6) were serum starved and treated with DCF-DA and growth factors, either in the presence or absence of N-acetyl cysteine or DPI. Two min after the addition of growth factors, total fluorescence was determined using a fluorescence plate reader at excitation and emission wavelengths of 485 and 530 nm, respectively. Values of DCF relative fluorescence units (RFU) measured from untreated and growth factor stimulated cells were evaluated for statistical significance by the Student t-test. Figure 5 depicts the production of ROS induced by different growth factors in human lens epithelial cells. EGF, FGF, TGF-β, PDGF, and lysophosphatidic acid (LPA) augmented production of ROS compared to untreated control samples (by 55%, 46%, 41%, 30%, and 23% over untreated controls, respectively, p<0.05). On the other hand, pretreatment of cells with either with N-acetyl cysteine or DPI not only abolished growth factor mediated stimulation of ROS production, but also appeared to suppress basal levels of ROS production, as evidenced by the lower values of DCF fluorescence compared to untreated controls. N-acetyl cysteine consistently inhibited DCF fluorescence to a greater extent than DPI, suggesting the participation of other oxidative cellular mechanisms in addition to NADPH oxidase, in the generation of ROS in lens epithelial cells Figure 5. DPI treatment also consistently inhibited DCF fluorescence compared to untreated samples, indicating the existence of an endogenous ROS-producing NADPH oxidase activity in untreated cells. Intriguingly, there seems to be a large degree of variation with respect to the relative effects of DPI and N-acetyl cysteine in suppressing the ROS generation in growth factor treated samples. While these inhibitors exerted fairly consistent effects with respect to EFG, FGF, and TGF-β, the effects were much stronger in the case of LPA and PDGF. Some of this variability could conceivably be attributed to differences arising from batch-to-batch variability between different cell populations, in terms of basal ROS production. Additionally, the ROS detection assay also showed a large inter-assay variability.

Expression of LMW-PTPase in lens

In this study we have also profiled the expression of low-molecular weight protein tyrosine phosphatase in lens tissue. LMW-PTPase is a ROS sensitive enzyme that plays a critical role in growth factor-induced cell proliferation and cytoskeletal changes by regulating the phosphorylation status of growth factor receptors [35-38]. The activity of LMW-PTPase has been shown to be regulated by thiol-redox cycling via intercovertible conformational switching between active and inactive states, in the presence of ROS and glutathione [37]. To obtain evidence for the presence of this redox sensitive phosphatase in lens tissue, cDNA libraries derived from human lens were screened by PCR analysis using sequence-specific oligonucleotide primers. Figure 6 demonstrates the expression of LMW-PTPase in human lens cDNA libraries and in a human lens epithelial cell line.

Discussion

The results of this study provide evidence for the expression and activity of a nonphagocytic NADPH oxidase system in lens tissue from several different species. We have characterized this lens NADPH-oxidase activity using several independent approaches including enzymatic assays, immunological assays, and PCR/RT-PCR analysis. Furthermore, the ROS-producing potential of this NADPH oxidase activity appears to be stimulated in response to growth factors in human lens epithelial cells. To our knowledge this is the first study to report a functionally active nonphagocytic NADPH oxidase system in lens tissue.

The nonphagocytic NADPH oxidase is constitutively active, but produces low levels of O_2^- and oxidants intracellularly [14,15]. This system appears to be present in various cell types including endothelial and epithelial cells of different tissues [14-21]. Oxidants generated by this enzyme are thought to have an important role in redox signaling, especially in nonphagocytic cells [23-27]. Although oxidants are typically considered deleterious to cell survival through protein oxidation, low levels of ROS produced in a regulated fashion have been shown to influence various cellular processes including proliferation, differentiation, cell cycle progression, cytoskeletal organization, and cell adhesion [14-21]. Additionally, redox dependent post-translational modification of proteins displaying a wide array of activities is emerging as a key signaling system that has been conserved throughout evolution and that influences many aspects of cellular homeostasis [26,36,38,39].

Signal transduction by oxygen species through reversible phosphotyrosine phosphatase inhibition, represents a widespread and conserved component of the biochemical machinery that is triggered by RTKs [22-24,39,40]. Various growth factors have been demonstrated to influence the production of ROS, and the Rac GTPase is considered to have a decisive role in this response [22-25,34,36,41-43]. We previously demonstrated the growth factor-stimulated activation of Rho and Rac GTPases in lens epithelial cells, and also that under these conditions, lens cells exhibited a dramatic reorganization of the actin cytoskeleton [7]. Several cell-surface receptors including PDGFR and EGFR, whose engagement has been shown to result in rearrangment of the actin cytoskeleton, trigger the generation of ROS in a Rac-dependent fashion [38]. Furthermore, LMW-PTPase has been found to be a direct target for Rac-mediated ROS production [36-38].

Towards the goal of further understanding the potential role(s) of Rac GTPase in lens and in lens epithelial cell function, the Rac-associated NADPH oxidase system was characterized in this study. Rac GTPase has been demonstrated to play a critical role in the regulation of NADPH oxidase-catalyzed generation of ROS in both phagocytic and non-phagocytic cells [8,9]. The data derived from lucigenin chemiluminescence of lens homogenates obtained from different species in the presence and absence of SOD and DPI (the flavoprotein inhibitor) confirms the presence of an enzymatically active NADPH dependent superoxide generating system in lens tissue (Figure 1). Further, the immunological evidence, (Figure 3) obtained for the presence of certain components (p67phox and p47 phox) of the NADPH oxidase system, together with molecular evidence obtained from PCR and RT-PCR analyses (Figure 2) of the expression of cytosolic (p67phox, p40 phox, and Rac1) and cytochrome b558 subunits (p22phox and p91 phox) strongly suggest the constitutive expression of an NADPH oxidase system in lens tissue. Additionally, lenses obtained from different species including monkey, bovine, rat, and human showed detectable levels of NADPH oxidase activity, as determined by the lucigenin assay. The production of superoxide anion by NADPH oxidase involves assembly of membrane-bound and cytosolic components to generate an activated functionally competent form of this enzyme [12]. Therefore, although the p67phox and p47 phox are known to be cytosolic, they could be distributed in the cytosolic and membrane fractions, as evidenced by the positive immunoreactivity of both soluble and insoluble lens fractions, for p67phox and p47 phox (Figure 3).

To obtain further insight into the significance of NADPH oxidase activity in lens, we decided to explore the effects of growth factors on ROS production. For this human lens epithelial cells were treated with different growth factors including EGF, FGF, TGF-β, PDGF, and LPA. These studies convincingly demonstrated the ability of the growth factors tested to increase levels of ROS production in an anti-oxidant (N-acetyl cysteine) and DPI-sensitive fashion. Both these agents inhibited the enhanced production of ROS induced by growth factors, confirming the participation of an NADPH oxidase system. Interestingly, both N-acetyl cysteine and DPI not only suppressed the ROS produced in response to growth factors, but also suppressed DCF fluorescence to levels lower than those observed in control cells (Figure 5). This effect of N-acetyl cysteine and DPI on lens epithelial cells suggests two possibilities. One is that there seems to be some basal level of flavoprotein-dependent ROS production in lens epithelial cells, and the second is that there might be other cellular mechanisms (flavoprotein-independent) participating in the production of ROS in lens epithelial cells. Furthermore, the variability between the experiments or between the growth factors with respect to the effects of N-acetyl cysteine and DPI suggest that there is a considerable variation in the levels of ROS generating activity between the different batches of cells used in this study. However, in human lens epithelial cells, there seems to be a definite stimulation of ROS production induced by different growth factors. Interestingly, EGF induced maximum ROS generation and in our previous study, we have also found maximum activation of Rac by EGF in human lens epithelial cells [7]. EGF also has been demonstrated to produce ROS in Rac GTPase dependent fashion in other cell type [25].

Low molecular weight protein-tyrosin phosphatase (LMW-PTPase) is an enzyme involved in mitogenic signaling and cytoskeletal rearrangement in response to PDGF stimulation [36-38,44]. LMW-PTPase is oxidized and inactivated both in vitro and in vivo by exogeneous oxidative stress and by endogeneously generated oxidative burst produced during PDGF signaling [36]. It has been characterized as an important target of redox signaling pathways and participates in growth inhibition and differentiation [45]. Its expression was determined in lens tissue to identify potential targets of the ROS generating system in the lens. Human lenses and human lens epithelial cells exhibited readily detectable levels of LMW-PTPase expression by PCR and RT-PCR analysis (Figure 6). Interestingly, lens tissues have also been reported to exhibit high levels of expression of an 18 kDa protein tyrosine phosphatase whose molecular weight matches that of LMW-PTPase [46]. Furthermore, ROS have been shown to down regulate Rho GTPase activity through inhibition of LMW-PTPase, thereby leading to increased tyrosine phosphorylation and activation of p190Rho-GAP [38]. Lens tissue expresses p190Rho-GAP, another well-characterized LMW-PTPase substrate (unpublished data). Previously we have documented the effects of growth factor stimulation on cytoskeletal organization in lens cells [7], and in this study. We showed increased production of ROS by growth factors. The effects of growth factors on generation of ROS in lens epithelial cells, when viewed in light of our earlier data on growth factor triggered cytoskeletal reorganization in such cells, indicate that activation of redox signaling pathways likely links these cellular events.

The ocular lens is a transparent tissue which contains high concentrations of soluble protein. Protein solubility and aggregation can be influenced by oxidative stress, and increased oxidative stress is thought to have deleterious effects on maintenance of lens transparency, leading to cataract formation [30,31]. Therefore, if ROS generation is not in homeostasis, lens function could potentially be compromised. Regulated production of ROS may, however, very well participate in normal lens growth and function through redox signaling, as has been shown for several cellular processes [23-27]. Additionally, a study by Chen et al. [47] has recently demonstrated the effects of redox signaling on the activation of MAP kinases in lens epithelial cells, while Li et al. [48,49] have reported the effects of H₂O₂ on c-myc and c-fos expression in lens epithelial cells, providing evidence for the participation of redox signaling in the mitogenic response.

In conclusion, we have shown that lens tissue expresses different components of NADPH oxidase and contains a functionally active ROS producing system. This enzyme activity appears to be augmented by external factors such as growth factors. Based on the effects of growth factors on ROS production, it is reasonable to suggest that ROS may play an important role in lens growth and function via their ability to regulate redox-signaling pathways in lens tissues. The regulation of ROS production in lens might be a very important aspect that needs to be explored in further studies.

Acknowledgements

We wish to thank Drs. V.N. Reddy, Tom Leto, Toshi Shinora and Pedro Gonzalez for providing human lens epithelial cell line, various antibodies to the different components of the NADPH oxidase and human lens cDNA libraries, respectively. Funded by research grants EY 12201 and EY 13573 from the National Eye Institute, NIH.

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