Coordinate Signaling by Src and p38 Kinases in the Induction of Cortical Cataracts

Abstract

purpose. The goals of this study were to determine whether MAP kinase signaling pathways play a role in the formation of lens cataracts and to examine the potential signaling relationship between Src and MAP kinases in signaling the induction of lens opacities.

methods. Embryonic day (E)10 chick lenses were cultured in Medium 199 containing 10% fetal bovine serum. The activation state of Src kinases and the MAP kinases extracellular signal-regulated protein kinase (ERK), c-jun N-terminal kinase (JNK), and p38 in the lens epithelium was determined over a time course from 10 minutes to 10 days in culture by immunoblot analysis. Src kinase activation was suppressed by exposure to the Src family kinase-specific inhibitor PP1. To examine the role of specific MAP kinases in the development of lens opacities, lenses were grown for 10 days in the presence or absence of inhibitors of ERK (U0126), JNK (SP600125), and p38 (SB203580). Lenses were observed and photographed daily, and the degree of opacification was quantified by using image-analysis software.

results. Within a short time after placing embryonic lenses in culture conditions that induce the formation of cataracts, there occurred a great increase in the activation state of the MAP kinase ERK. Activation of ERK was both rapid and transient. No activation of the MAP kinase JNK was observed in the cataract cultures beyond that which occurred in normal lens epithelium, even though JNK activation is often linked to the cellular response to stress. In contrast, although p38 activation was barely detected in the normal embryonic lens, this stress-activated protein kinase exhibited a robust activation in cataract cultures that was sustained throughout the culture period. Studies conducted to map the cataract signaling pathways indicate that the p38 MAP kinase functions upstream of the Src kinase. To analyze the potential role of ERK, JNK, and p38 in cataract induction, lenses were cultured in the presence of specific MAP kinase inhibitors. Although the inhibitors of ERK and JNK did not interfere with the formation of cataract, p38 inhibitors blocked the development of lens opacities with an efficacy similar to that of the Src kinase inhibitor PP1.

conclusions. Activation of both Src and p38 kinases lead to the induction of cataract.

Cataract is a multifactorial disease. Many stress factors, including oxidative stress, ^{1 2 3} osmotic stress, ^{4 5 6} and UV light ^{7 8 9 10} can induce formation of cataracts, but the signaling pathways involved in the induction of cataract by stress are poorly understood. The Src family tyrosine kinases stand out as signaling intermediates common to pathways activated by many distinct stress factors. Src kinases have been shown to be involved in signaling events stimulated by reactive oxygen species, osmotic stress, UV light, and extracellular mechanical stress, in several different cell types. ^{11 12 13 14 15 16} We have shown that Src kinase activation is central to the induction of lens cataracts. ¹⁷ In these studies the activation of Src kinase signaling pathways in cultured chick embryo lenses leads to an imbalance in normal cellular function so that cataracts form. The development of opacities is blocked when the embryonic lenses are cultured the presence of specific inhibitors of Src family kinases. 

The MAP kinases—extracellular signal-regulated protein kinase (ERK), c-jun N-terminal kinase (JNK), and p38 kinase—are key elements in signal-transduction pathways induced by extracellular stimuli. ^{18 19 20} Like the Src kinases, the activation of MAP kinases is characteristic of the cellular response to many distinct stress inducers. Oxidants, radiation, and high glucose can activate ERK, ^{21 22 23} JNK, ^{21 24 25 26 27} and p38 kinases. ^{21 28 29 30} The MAP kinase signaling response in a cell can be specific to the particular extracellular stimulus. Distinct responses have been observed for different growth factors, cytokines, and cellular stresses. ^{25 30 31} This has been seen in the signaling response of lens epithelial cells to different stress factors. In the epithelium of cultured lenses, hyperglycemia-induced osmotic stress activates p38 but not JNK, whereas bFGF activates JNK but not p38. Both of these stimuli activate the Raf-MEK-ERK pathway. ³² In HLE cells, a human lens epithelial cell line, UV B and UV C radiation activate p38 and JNK, but not ERK. ³³ Stress factors work through the different MAP kinases to regulate cell proliferation, differentiation, cell structural reorganization, and cell death. ^{34 35 36 37 38}  

Activation of MAP kinases also has been shown to induce formation of cataract. Cataracts develop in transgenic mice that overexpress MEK1, an upstream activator of ERK, through a mechanism that involves abnormalities in glucose metabolism that damage the lens fiber cells. ³⁹ In the cataractous lenses of diabetic rats, ERK, p38, and JNK activities are upregulated. ⁴⁰ These studies suggest that the upregulation of MAP kinases in the lens by external stimuli may serve as a common pathway in cataract induction. 

The activation of MAP kinases by extracellular stresses such as UV light, hydrogen peroxide, high osmolarity, and shear stress involves the activation of Src kinases. ^{14 16 22 41 42} Therefore, in the present study, we investigated the role of MAP kinases in the Src signaling pathway that leads to the formation of cataract in our lens organ culture model. We hypothesized that extracellular stresses produced in the cataract cultures induce the formation of lens opacities through a common pathway that involves both Src and MAP kinases. We have determined which MAP kinases are activated in the lens cultures, their temporal pattern of activation, and the hierarchy of Src and MAP kinase signaling. Moreover, by using specific MAP kinase inhibitors, we examined which of the MAP kinases played a central role in the induction of lens cataract. Our results demonstrate that both ERK and p38 are activated in the epithelium of lenses grown in cataract-inducing culture conditions. Whereas ERK induction was transient, the activation of the p38 kinase was sustained and coincident with the formation of cataracts. Inhibitors of p38 suppressed the activation of Src and blocked the formation of lens cataracts. 

Materials and Methods

Lens Organ Culture

The lens organ culture system was prepared as reported previously. ¹⁷ Briefly, embryonic day (E)10 chicken lenses were isolated from the surrounding eye tissue and any associated vitreous or ciliary body removed. Tissue collection conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The embryonic lenses were placed one to a well in 24-well tissue culture dishes in the presence of Medium 199 (Invitrogen-Gibco, Gaithersburg, MD) containing 10% fetal bovine serum (Invitrogen-Gibco), 0.1 μg/mL l-glutamine, and penicillin-streptomycin, in an atmosphere of 5% CO₂ and 95% air. The cultured lenses were treated with the following inhibitors: the Src inhibitor PP1 (Biomol, Plymouth Meeting, PA), the ERK inhibitors U0126 or PD98059 (Biomol), the JNK inhibitor SP600125 (Biomol), and the p38 inhibitors SB203580 or SB202190 (Biomol and Calbiochem, La Jolla, CA). Dimethyl sulfoxide (DMSO), the vehicle for the inhibitors, was added to the control lens cultures. Culture media containing either the inhibitors or DMSO alone were renewed every second day throughout the culture period. 

Quantification of Lens Opacity

Lenses were observed and photographed every second day in culture. The total area of each lens and the area of opacification were determined using phase 3 image-analysis software (Phase 3 Imaging Systems, Glenn Mills, PA), after which the percentage of the lens area that had become opaque was determined. 

Tissue Extraction and Protein Determination

Before analysis, the entire lens epithelium, which contains both the anterior and equatorial epithelia, was removed by microdissection from the underlying fiber cells of the cultured lenses. The isolated lens tissue fractions were extracted in OG buffer (1% Triton X-100, 44.4 mM n-octyl β-d glucopyranoside in 100 mM NaCl, 1 mM MgCl₂, 5 mM EDTA, and 10 mM imidazole) containing the following inhibitor cocktail: 3 mM sodium pyrophosphate, 50 mM sodium fluoride, 50 μg/mL aprotinin, 25 μg/mL soybean trypsin inhibitor, 100 mM benzamidine, 5 μg/mL leupeptin, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM sodium vanadate, and 0.2 mM H₂O₂. Protein concentration was determined by the bicinchoninic acid (BCA) assay (Pierce, Rockford, IL). 

Antibodies

Antibodies used in this study included activated Src (phospho-Src⁴¹⁸, Biosource, Camarillo, CA), ERK1/2 and phospho-ERK1/2 (Promega, Madison, WI), phospho-JNK (Biosource and Promega, Madison, WI), phospho-p38 (Promega), c-Src and p38 (Santa Cruz Biotechnology, Santa Cruz, CA), and JNK1/2 (Upstate Biotechnology, Lake Placid, NY). Horseradish peroxidase (HRP)–conjugated antibodies were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA). 

Immunoblot Technique

Equal amounts of total cellular protein (10 or 15μg) were separated on Tris-glycine gels (Novex, San Diego, CA), electrophoretically transferred to a membrane (Immobilon-P; Millipore Corp., Bedford, MA) and immunoblotted as described previously. ⁴³ Densitometric analysis was performed using Kodak 1D software (Eastman Kodak Company, Rochester, NY). All gels were run under reducing conditions. 

Preparation of Phospho-p38 Control

Primary quail lens cell cultures were prepared as previously described. ⁴⁴ Cells were plated at approximately 5 × 10² cells/mm² on 35-mm dishes coated with laminin (BD Biosciences, San Diego, CA) in complete lens medium (Medium 199; Invitrogen-Gibco) containing 10% fetal bovine serum (Invitrogen-Gibco). Primary lens cells, at 14 days in culture, were treated with 10 μM anisomycin (Sigma, St. Louis, MO) for 45 minutes to induce apoptosis. Cells were washed with PBS, harvested, and protein was extracted with OG buffer. 

Statistical Analysis

The Student’s t-test was used to determine whether there was a significant difference between two means (P < 0.05). 

Results

High Level Src Activation Sustained in Cataract Cultures

Our previous studies have shown that chick embryo lenses grown in organ culture develop cortical opacities covering approximately 25% of the lens area by 6 days in culture. ¹⁷ Formation of these cataracts is dependent on the signaling activity of Src family kinases. When these lenses are grown in culture in the presence of the selective Src kinase inhibitor PP1, they remain clear. ¹⁷ To examine the relationship between the activation state of Src kinases in the lens epithelium and the formation of cortical cataract, we determined the temporal pattern of Src kinase activation in the absence and presence of the Src kinase inhibitor PP1. Activation of Src was measured by immunoblot analysis using an antibody to the phosphorylated tyrosine in its active domain (Src Y⁴¹⁸). For this analysis, as throughout this study, we examined activation of the kinase at two distinct time periods in culture. To determine the rapidity of the signaling response and the possibility that this response was transient, kinase activation was examined at time points beginning at 10 minutes and extending through the first day in culture (Fig. 1A) . To investigate the possibility of a sustained signaling response we examined lenses at later culture times, between 1 and 10 days in culture, which includes the period during which cataracts develop (Fig. 1B) . 

Because the lenses are placed in organ culture at embryonic day 10, the epithelia of E10 lenses were used as the control. A high level of Src kinase activation was observed in these control epithelia (Fig. 1A) . This was not surprising, because Src kinases are well-known for their signaling role in cell proliferation and differentiation, ^{45 46 47 48 49 50} both of which are characteristics of the dynamic region of lens equatorial epithelium. Placing the E10 embryonic lenses in culture induced a rapid but transient increase (less than twofold) in the activation state of Src kinases (Figs. 1A 1C) , which returned to control levels within 18 hours. During the principle period of cataract formation, from 5 to 7 days in culture, the activation of Src kinases in the lens epithelium was as high as occurs at E10 (Figs. 1B 1C ; P > 0.05). This persistently high level of Src kinase activation both before and during cataract formation underlies the important role of the Src kinases in the signaling pathways that induce development of lens opacities. Similarity in the activation state of Src kinases in cataract cultures to that in embryonic tissue suggests that Src kinases are involved in functionally distinct signaling pathways during lens development and disease. 

Exposure to the Src kinase inhibitor PP1 at a concentration of 10 μM effectively attenuated the activation of Src kinases in the epithelium of the cultured lenses, with the level of Src Y⁴¹⁸ phosphorylation dropping to less than 10% of the control level during the first day in culture (Figs. 1A 1C) . At this concentration the PP1 inhibitor has been shown to be specific for the Src kinases. The activation state of Src kinases in the presence of PP1 remained low throughout the remainder of the culture period, and Src activation was barely detectable by day 7 of inhibitor treatment (Figs. 1B 1C) . The changes in Y⁴¹⁸ phosphorylation reflected a change in Src kinase activity, not in Src kinase expression (Figs. 1A 1B) . These results demonstrate that in the cultured lenses, the Src inhibitor PP1 effectively attenuated the autophosphorylation of Y⁴¹⁸ in the Src kinase active domain, thereby blocking the activation of Src, and preventing formation of cataracts. 

Effect of Glucose Concentration on Src Kinase Activation and Cataract Formation

Src kinases, which signal cataract induction in our lens culture system, can be activated downstream of many different stress pathways. ^{11 12 13 14 15 16} We investigated whether the stresses incurred by the removal of the embryonic lenses from their surrounding vitreous and aqueous humors and placing them in culture induced the Src pathways that caused cataracts to form. This possibility was suggested by studies that show disturbing the interaction between the vitreous body and the lens contributes to the development of cataract as well as affects the ability of the human lens to withstand metabolic stress. ^{51 52 53} Embryonic cells have a high rate of metabolism, and many chick primary culture systems, including myoblasts and fibroblasts, grow preferentially in high-glucose medium. ^{54 55} We hypothesized that cataracts form in our culture system because the low glucose concentration in Medium 199 does not support the metabolic needs of the embryonic lenses after they are removed for culture. 

To test this hypothesis we increased the glucose concentration of our culture medium to 14 mM, similar to that of the high-glucose medium used for culturing embryo-derived cells. As predicted, increasing the glucose concentration to 14 mM prevented the formation of cortical cataract (Figs. 2A 2B) . On further analysis, we found that even smaller increases in glucose concentration could attenuate cataract formation (data not shown). To examine whether raising the glucose concentration in our culture medium prevented formation of cortical opacities by blocking a stress-induced Src pathway, we examined the level of activation of Src kinases in high-glucose medium. At 14 mM glucose, Src kinase activation (Y⁴¹⁸ phosphorylation) was attenuated with an efficacy similar to that of the Src kinase-specific inhibitor PP1 (Fig. 2C) , suggesting that the increased glucose concentration prevents cataract formation by blocking a Src kinase signaling pathway. 

Src Kinase–Independent Transient Activation of the MAP Kinase ERK in Lens Cataract Cultures

A typical signaling response of cells exposed to stress is activation of the MAP kinase ERK. ^{56 57} Therefore, we examined whether activation of ERK was increased when chick embryo lenses were placed in culture conditions that induce the formation of lens opacities. ERK activation in the lens epithelium was determined by immunoblot analysis at multiple time points during the 10-day culture period. The epithelia of normal E10 lenses were used in control experiments, because we have shown ERK to be expressed and activated in the embryonic lens. ⁵⁸ At only 10 minutes in culture there was a dramatic increase in the activation state of ERK, with ERK activation reaching levels four times greater than in control lens epithelia (Figs. 3A 3C) . The activation of ERK in culture was transient, returning to control levels within 4 hours. ERK activation then dropped to a lower baseline and remained low for the duration of the culture period (Figs. 3B 3C) . ERK expression itself was not affected by the lens culture conditions (Figs. 3A 3B) . Because cataracts are first observed in the lens cultures at day 5, ¹⁷ any correlation between the induction of ERK activation and the formation of lens cataracts is likely to be indirect. 

Src kinases have been implicated in signaling roles both upstream and downstream of MAP kinases. ^{14 16 22 41 42} Because activation of Src family kinases leads to induction of lens opacities, we examined whether the activation of the MAP kinase ERK in the lens cataract cultures was dependent on activation of Src kinases. The large spike in ERK activation observed immediately after lenses were placed in culture was not inhibited by the presence of the Src kinase inhibitor PP1 (Figs. 3A 3C) . However, at later times in culture when ERK signaling was low, the Src inhibitor PP1 had a small but significant negative effect on ERK activation (Figs. 3B 3C ; P < 0.05, 2d, 5d). 

Activation State of the Stress-Activated Kinases p38 and JNK in Lens Cataract Cultures

The MAP kinases JNK and p38, known as the stress-activated protein kinases, are activated by many different stress stimuli. ^{24 25 26 30} We examined whether either of these MAP kinases was activated in the cataract lens cultures with a temporal pattern consistent with their involvement in signaling the induction of lens opacities. Activation of JNK and p38 in the lens epithelium was determined by immunoblot analysis at multiple time points during the culture period. 

No change in the activation state of JNK1 or -2 was observed in the epithelium of lenses grown in culture conditions that induce cataract (data not shown). At each point examined throughout the 10-day culture period, activation of both JNK1 and -2 was at the same level as occurred in the normal E10 lens epithelium, but much below the level of JNK activation elicited by treatment of lens cells with anisomycin. These results indicate that it is unlikely that the JNK MAP kinase plays a signaling role in the formation of lens cataracts. 

In the epithelium of normal E10 lenses, activation of p38 could barely be detected, even though the protein was expressed (Fig. 4) . Note that the phospho-p38 antibody cross-reacted with phospho-ERK, which migrated just above phospho-p38 and gave a strong signal in the normal E10 lens epithelium. To demonstrate that this higher molecular weight band at E10 was phospho-ERK, we exposed the cultured lenses to the ERK inhibitor U0126 before analysis, which removed this band from the phospho-p38 blot (Fig. 4C) . Because it was difficult to detect activation of p38 in normal embryo lenses, we included primary lens cell cultures exposed to the apoptosis-inducer anisomycin ⁵⁹ as a positive control for activation of the p38 MAP kinase (Fig. 4A) . 

There was no immediate signaling response of p38 to the cataract culture conditions. Its activation state at 60 minutes was similar to that observed in the control lens epithelia (Fig. 4A) . This is the same time period in which ERK was highly activated (see also Fig. 3 ). Activation of p38 remained low throughout the first few hours in culture (data not shown). In contrast, by 1 day in culture, there was a robust activation of p38, with the activation state of p38 approaching that in anisomycin-treated lens cell cultures (Fig. 4A) . This high level of p38 activation was sustained throughout the 10-day culture period (Fig. 4B) . The activation of p38 preceded the appearance of lens opacities and p38 remained active throughout the time during which cataracts developed in the cultured lenses. Expression of p38 was unaffected by the culture conditions. 

Minimal Role for Src Kinases in the Activation of MAP Kinases in Lens Cataract Cultures

Exposure of the cultured lenses to the Src kinase inhibitor PP1 had little effect on the activation state of either ERK (Fig. 3) or JNK1 and -2 (data not shown). Similarly, there was no significant change observed in the activation state of p38 when the lenses were grown in culture in the presence of the Src kinase inhibitor PP1 (Fig. 5) . These results suggest that although activation of Src kinases leads to cataract induction, ¹⁷ Src kinase signaling is not positioned upstream of MAP kinase activation in the induction of lens cataract. 

Role of p38 MAP Kinase in the Activation of Src in Cataract Cultures

We next investigated whether activation of the MAP kinase p38 might be upstream of Src kinase activation in the cataract cultures. We focused these studies on the p38 kinase because it was the only MAP kinase whose activation was maintained throughout the cataract culture period. For these studies we determined whether the p38 inhibitor SB203580 would block the activation of Src kinases in the cataract cultures. At time points less than 1 day after the lenses were placed in the culture, the p38 inhibitor had only a small effect on the activation state of Src (Figs. 6A 6C) . This was not surprising, as we had already shown that the activation of p38 was minimal at these early culture times (Fig. 4A) . However, between days 2 and 5 in culture, when activation of p38 was high (Fig. 4B) , exposure of lenses to the p38 inhibitor suppressed the activation of Src kinases (Figs. 6B 6C ; P < 0.05). It is also during this time that opacification of the cultured lenses is first observed. ¹⁷ These results suggest that p38 is activated upstream of Src in the signaling pathway that induces lens opacities. 

p38-Src Signaling Pathway in the Induction of Lens Opacities

To determine whether the activation of MAP kinase signaling pathways in the lens cultures leads to the induction of lens opacities, E10 lenses were cultured in the presence of specific inhibitors of the MAP kinases ERK, JNK, and p38 for 10 days. Figure 7 shows the results of a dose–response study of each of the MAP kinase inhibitors examined. Inhibitor-treated lenses were compared with control cultures to which the vehicle DMSO had been added. The control lens cultures exhibited a degree of opacity of approximately 25%. Exposure of the cultured lenses to the ERK inhibitor U0126 had no effect on the degree of opacification (Fig. 7A) . The area of opacification in the presence of the ERK inhibitor at 10 days in culture was similar to that observed in untreated lenses in culture. The type of cataract that formed in culture also was unaffected by the presence of the ERK inhibitor. As in control lens cultures (DMSO), cultured lenses exposed to the ERK inhibitor U0126 showed development of cortical cataracts (Fig. 8) . Similar results were obtained with another ERK inhibitor, PD98059 (data not shown). 

The JNK inhibitor SP600125 also failed to block the formation of cataracts in the lens cultures, the area of opacity remained at approximately 23% (Fig. 7B) . In the presence of the JNK inhibitor the lenses formed cortical cataracts that appeared similar to those typically observed in the cataract cultures (Fig. 8) . No significant differences were found in the total area of opacification between lenses in the control cultures and lenses treated with either the ERK or JNK inhibitors (Figs. 7A 7B ; P > 0.05). 

In contrast, the p38 inhibitors SB203580 and SB202190 effectively suppressed the development of cortical opacities in the lens cultures (Figs. 7 8) . In a dose–response study we found that as the concentration of the p38 inhibitor increased from 0.1 to 10 μM, the degree of lens opacity decreased from 20% to 4% (Fig. 7C) . The lenses in cultures exposed to the p38 inhibitors remained strikingly clear, similar to lenses grown in the presence of the Src kinase inhibitor PP1 (Fig. 8) . 

The difference in opacification between lenses in the control cataract cultures and those grown in the presence of the p38 inhibitor was significant (Fig. 7C , P < 0.01). No difference was found between the degrees of opacification in the PP1 Src inhibitor–treated lenses and those treated with the p38 inhibitor (Fig. 7D , P > 0.05). Taken together, these studies suggest that activation of p38, but not ERK or JNK, leads to the formation of lens cataract. p38, a well-known stress-induced kinase, is likely to be a component of the Src signaling pathway that we have shown in earlier work to induce cataract formation. 

Discussion

Although there are numerous factors that cause cataracts to form, the signaling pathways that induce cataract formation are not well understood. In this study we investigated the involvement of classic stress factor–signaling molecules, the Src and MAP kinases, in the induction of lens opacities in our chick embryo lens cataract culture model and begun to map the cataract-signaling pathway. We have determined that Src kinase activation is essential for the induction of cataracts in this model system. ¹⁷ In these lens cataract cultures, cortical opacities are observed at day 5. The opacities continue to increase in severity during the culture period, but remain cortical. When the embryonic lenses are cultured in the presence of specific Src kinase inhibitors, cortical opacities fail to develop, and the lenses remain clear, thus demonstrating that activation of Src kinase-dependent–signaling pathways leads to the induction of lens cataracts. 

The phenotype of the cataracts that develop in this culture model is that of metabolic cataract. This conclusion is based on the ability to rescue the lenses from cataract by increasing the glucose concentration of the media to that found in high-glucose medium, an optimal culture environment for many embryonic cell types. Increasing the glucose concentration in the medium blocked cataract formation through a mechanism involving the suppression of Src kinase activity. The reason for the high-energy needs of these cultured lenses is unclear. The possibility that younger lenses, particularly embryonic lenses, may be more susceptible to cataract induction in culture is supported by studies in which cataract is induced by culturing lenses in serum-free conditions. Lenses from chick embryos remain transparent for just 3 hours, ⁶⁰ from weaned rats for 5 days, and from adult rats for 7 days. ⁶¹ It is also possible that cataracts form in our culture system because the low glucose concentration in Medium 199 does not support the increased metabolic needs of the embryonic lenses as they attempt to recover from the mechanical stress incurred by removing them for culture. 

Although these data suggest that the increased metabolic needs of embryonic cells is a contributing factor to the induction of cataract in our culture model, they fail to clarify whether these cataracts have any similarity to human cataract in vivo. Findings in recent studies in our laboratory aimed at understanding the mechanism of cataract induction in the lens organ cultures suggest that the cortical cataracts form in response to the apoptotic death of cells in the lens epithelium (Zhou J and Menko AS, manuscript in preparation). In fact, apoptosis in the lens epithelium is a well-documented characteristic of human cataracts and common to many cataract models, particularly those induced by stress. ^{62 63 64} Although the inductive signal in our lens cataract model may be specific to chick embryo lenses, we suggest that the activation of similar stress-induced signaling pathways is likely to be a cause of naturally occurring cataracts. 

Because attenuation of Src kinase activity blocks the development of lens opacities in our cataract culture model, we examined whether there was a temporal correlation between the activation state of Src kinases and the appearance of lens opacities. We found that not only was Src activation high in the epithelial cells of lenses in cataract culture, but that it was equally high in the epithelium of normal E10 lenses. These results suggest that Src kinases function through different pathways in lens development and disease. In development, Src kinases play a crucial role in the signaling events that regulate cell proliferation and differentiation. ^{46 48 49 50 65 66 67} through pathways intrinsic to both integrin and growth factor receptor pathways. ^{45 68 69 70 71} In the developing lens at E10, high levels of Src kinase activity are consistent with the function of the embryonic lens epithelium, which contains both a zone of proliferation and the region in which lens fiber cell differentiation is initiated. In the lens disease process that leads to cataract formation, the Src kinases must be activated by distinctly different stimuli than during development. The activation of Src under these conditions is likely to reflect their role as a common signaling effector of stress-activated pathways. ^{14 15} In fact, the high level of Src activation in the cataract cultures parallels the activation of many signaling molecules in cells exposed to stress. ^{11 13 16 72 73}  

Another consideration is that there are many different members of the family of Src kinases, including molecules such as Src, Fyn, Lyn, and Yes, ^{74 75} each with the potential for distinct cellular signaling functions. The processes of cell development and cell stress may activate different Src family kinase members. Regardless of which Src kinases are activated in the lens cultures, the presence of the Src inhibitor PP1 effectively inhibits most activation of the Src kinase by 1 day in culture. By 5 days in culture, when cataracts have developed in control cataract cultures, activation of Src kinase was barely detected in lenses kept clear by exposure to the PP1 Src kinase inhibitor. 

To further understand cataract signaling, we investigated the role of the MAP kinases in the induction of lens opacities. We chose to study the MAP kinases because members of this family of protein kinases function both as components of linear Src signaling pathways and in coordinate signaling pathways with the Src kinases. ^{12 14 22 41 42} The response of the ERK, JNK, and p38 MAP kinases to the culture conditions was distinct. Only ERK exhibited an immediate response when the lenses were placed in culture, but this increase in ERK activity was transient. By 4 hours in culture ERK activation was lower than controls and remained low throughout the 10-day culture time. The low level of ERK activation throughout the majority of the culture time suggested that ERK signaling was not responsible for cataract induction. Indeed, when the lenses were cultured in the presence of ERK inhibitors, they failed to prevent formation of cataracts in this model. 

The induction of JNK activation has been associated with many distinct and sometimes opposing cell-signaling pathways. For example, JNK is involved in both the induction and the prevention of apoptosis. ^{34 76} In the cataract-inducing culture system, however, there was no change in the activation state of JNK from that in the epithelia of day 10 embryonic lenses. In both E10 lenses and lenses growing in cataract-inducing conditions, the activation state of JNK was low compared with lens cell cultures exposed to the apoptosis-inducing agent anisomycin (data not shown). From these results, we conclude that JNK is unlikely to play an inductive role in the formation of cataract. In support of this conclusion, we found that when the lens cultures were grown in the presence of an inhibitor of JNK, there was no effect on the ability of the lenses to form cataracts. 

p38 is another stress-activated protein kinase. The epithelial cells of the embryonic lens express p38, but in contrast to ERK and JNK, only very low levels of p38 kinase activation were detected in the E10 lens. Robust activation of p38 did not occur until 1 day in culture. This high level of p38 activation persisted throughout the period of cataract development and was similar to that observed in lens cell cultures induced to undergo apoptosis by exposure to anisomycin. p38 appears to be the only MAP kinase to function as a signaling intermediate in the pathways leading to cataract induction. In support of this conclusion, inhibitors of p38 effectively suppressed the development of cataracts, keeping the lenses distinctly clear much like the result we have previously obtained for lenses exposed to the Src kinase inhibitor PP1. 

We have mapped the relationship between the Src and p38 kinases to determine how they may coordinate signaling of cataract induction. To examine the direction of Src and p38 signaling in the lens cataract-inducing cultures we examined the effect of inhibitors of each kinase on the activation state of the other. We had expected that the Src kinase would function upstream of p38. We found unexpectedly that inhibiting the activation of Src kinase had little effect on the activation state of p38. In contrast, at times in culture preceding the appearance of lens opacities, the p38 inhibitor had a significant inhibitory effect on the activation state of Src. These results suggest that in cataract-inducing conditions, p38 signaling is upstream of the activation of Src. 

Is there a temporal relationship between the activation of p38 and Src kinases and the development of lens cataracts? Although both Src and p38 kinase activities are high for several days before the appearance of cortical opacities, the requirement for a p38 signal to maintain maximum activation of Src kinases is most pronounced in the days just preceding the appearance of cataracts. This is exactly the period that one would predict to be crucial to the induction of cataract in this lens culture system. These results suggest that there is a unique cataract-inducing Src kinase signal that is specifically regulated by a p38 kinase-signaling pathway. We hypothesize that p38-Src signaling pathways are likely to be common to many distinct cataract-inducing factors. 

 Supported by National Eye Institute Grants EY014258 and EY10577 (ASM) and an ARVO/Alcon Laboratories, Incorporated Research Fellowship Grant (JZ).

 Submitted for publication November 11, 2003; revised March 4, 2004; accepted March 23, 2004.

 Disclosure: J. Zhou, None; A.S. Menko, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Figure 1.

 

Sustained high level of Src activation in cataract-inducing cultures. E10 chick embryo lenses were grown under culture conditions that induced formation of cataract in the absence and presence of the Src kinase-specific inhibitor PP1. Lens epithelial cells were monitored for the activation state of Src family kinases throughout a 10-day culture period. Immunoblot analysis for active Src kinases was performed with an antibody to the phosphorylated tyrosine at position 418 of the kinase domain (pSrc⁴¹⁸). Total Src expression also was determined at each time point examined (Src). (A) Early effects of culture on Src kinase activation. (B) Sustained, long-term effects on Src during the 10-day culture period. E10, epithelial cells isolated directly from the E10 lens. (C) Src activation was quantified after densitometric scanning of immunoblots from four independent experiments and data were normalized to E10. Activation of Src kinases was high in normal E10 lens epithelium. Culture conditions induced a rapid but transient increase in Src activation. Src activation was sustained at high levels throughout the culture period. PP1 effectively attenuated Src activation within 10 minutes, and Src activation remained low in the presence of PP1.

Figure 2.

 

Increased glucose concentration blocked cataract formation by attenuation of Src kinases. E10 chick embryo lenses were grown in normal culture medium or high-glucose medium in which the glucose concentration was raised to 14 mM. At day 0 and day 10 in culture, (A) lenses were photographed and (B) the area of opacification determined. (C) The activation state of Src kinases was determined at 1 hour by immunoblot analysis using an antibody to phosphorylated Y⁴¹⁸ in the Src kinase active domain. Total Src expression also was determined. Lenses cultured in normal and high-glucose medium were compared with those treated with the Src inhibitor PP1. Increasing the glucose concentration of the lens culture medium to 14 mM blocked the development of lens opacities by suppressing a Src-kinase signaling pathway. Note that both 14 mM glucose and PP1 suppressed Src kinase activity despite an increase in Src expression.

Figure 3.

 

Rapid but transient activation of the MAP kinase ERK in the cataract cultures. ERK activation in epithelial cells isolated from lenses grown in cataract-inducing culture conditions in the absence and presence of the Src kinase-specific inhibitor PP1 was determined in both (A) lenses less than 1 day in culture and (B) lenses at 1 to 10 days in culture. The activation state of ERK was determined by immunoblot analysis with an antibody to phosphoERK (p-ERK). E10 lens epithelium served as the control. Total ERK expression (ERK) also was determined. (C) ERK activation was quantified after densitometric scanning of immunoblots from three independent experiments, and data were normalized to E10. In the lens cultures there was an early, transient activation of ERK that was unaffected by inhibition of Src kinases.

Figure 4.

 

Sustained activation of p38 in the cataract cultures. Activation of the stress-activated MAP kinase p38 was determined for lens epithelial cells isolated from lenses grown in cataract-inducing culture conditions. p38 activation was examined at (A) time points during the first day in culture and (B) time points throughout the remainder of the 10-day culture period. Total p38 expression also was determined at each time point. Activation of p38 was determined by immunoblot analysis with an antibody to phospho-p38 (p-p38). This antibody cross-reacted with activated ERK (p-ERK), which migrated just above activated p38, as confirmed by the disappearance of the p-ERK band when the lens cultures were grown in the presence of the ERK inhibitor U0126 (C). p38 activation was hardly detectable in the epithelium of normal E10 lenses (E10), the major band in this sample corresponded to p-ERK (A). At 10 minutes in culture, when p-ERK is highly activated, p38 remains nonresponsive. p38 activation at 1 hour in culture was similar to the E10 control. By 1 day in culture activation of p38 was robust and reached the same level of activation observed in lens cell cultures that were treated with an inducer of apoptosis, anisomycin (An). p38 activation remained high throughout the 10-day culture period (B). Blots are representative of three independent experiments.

Figure 5.

 

p38 activation in the presence of the Src inhibitor PP1. E10 lenses were cultured under conditions that induce cataract in the presence and absence of the Src kinase inhibitor PP1. p38 activation was determined for the lens epithelial cells by immunoblot analysis with an antibody to phospho-p38 (p-p38). This antibody cross-reacted with activated ERK (p-ERK), which migrated just above activated p38. Total p38 expression (p38) also was determined. (A) Lenses were examined at times in culture later than day 1, when activation of p38 was robust. E10, epithelial cells isolated directly from the embryonic day 10 lens. (B) p38 activation was quantified after densitometric scanning of immunoblots from three independent experiments, and data were normalized to day 2.

Figure 6.

 

Src activation was suppressed when p38 activation was inhibited. E10 lenses were grown in cataract-inducing conditions in the presence of the p38 inhibitor SB203580 and Src activation determined for lens epithelial cells by immunoblot analysis with an antibody to phospho-Src (p-Src418). Src expression (Src) also was determined. Lenses were examined both (A) at multiple time points during the first day of culture and (B) during a period of robust p38 activation in culture, between days 1 and 7. E10, epithelial cells isolated directly from the embryonic day 10 lens. (C) p38 activation was quantified following densitometric scanning of immunoblots from three independent experiments and data were normalized to E10.

Figure 7.

 

The p38 inhibitor has similar effectiveness to the Src inhibitor at preventing the development of cataracts. E10 lenses were grown in cataract-inducing culture conditions in the presence of (A) the ERK inhibitor U0126, (B) the JNK inhibitor SP600125, or (C) the p38 inhibitor SB203580. The area of opacification was determined at day 10 in culture. A dose–response analysis was performed for each inhibitor (A– C). (D) Comparison of E10 lenses grown in cataract-inducing culture conditions in the presence of the ERK inhibitor U0126, the JNK inhibitor SP600125, the p38 inhibitor SB203580, the Src inhibitor PP1, or their vehicle DMSO. Each inhibitor was used at a concentration of 10 μM. The area of opacification was determined at day 10 in culture. Only the p38 and Src inhibitors had a significant inhibitory effect on the formation of cortical cataracts.

Figure 8.

 

Inhibitors of the p38 MAP kinase block cataract formation. E10 lenses were grown in cataract-inducing culture conditions for 10 days in the presence or absence of inhibitors to either MAP or Src kinases. Control for these cultures was DMSO, the vehicle for all the inhibitors. These studies included the JNK inhibitor SP600125, the ERK inhibitor U0126, the p38 inhibitors SB203580 and SB202190, and the Src inhibitor PP1. All inhibitors were used at a concentration of 10 μM. Lenses were observed for the development of opacities at culture days 0, 6, and 10. All observed cataracts were cortical. Among the MAP kinase inhibitors, only p38 inhibitors blocked the formation of cataracts. Lenses treated with the p38 inhibitors appeared similar to those grown in the presence of the Src inhibitor PP1. The lenses depicted are representative of a minimum of 50 observed lenses.

The authors thank Marilyn Woolkalis and Gregory Weber for critical reading of the manuscript and Gregory Weber for providing anisomycin-treated lens cell cultures.