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Published as doi: 10.1096/fj.07-095455.
 (The FASEB Journal. 2008;22:2498-2507.)
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TGFβ mediates activation of transglutaminase 2 in response to oxidative stress that leads to protein aggregation

Dong-Myung Shin*, Ju-Hong Jeon{dagger}, Chai-Wan Kim*, Sung-Yup Cho*, Hye-Jin Lee*, Gi-Yong Jang*, Eui Man Jeong*, Dong-Sup Lee{ddagger}, Ja-Heon Kang§, Gerry Melino||, Sang-Chul Park* and In-Gyu Kim*,1

* Department of Biochemistry and Molecular Biology/Aging and Apoptosis Research Center (AARC),

{dagger} Department of Physiology and Biophysics, and

{ddagger} Cancer Research Institute, Seoul National University College of Medicine, Seoul Korea;

§ Department of Ophthalmology, Kyung Hee University College of Medicine, Seoul, Korea; and

|| Department of Experimental Medicine, University of Rome Tor Vergata, Rome, Italy

1Correspondence: Department of Biochemistry and Molecular Biology/AARC, Seoul National University College of Medicine, 28 Yongon Dong, Chongno Gu, Seoul 110–799, Korea. E-mail: igkim{at}plaza.snu.ac.kr


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Transglutaminase 2 (TGase2) is a ubiquitously expressed enzyme that catalyzes irreversible post-translational modification of protein, forming cross-linked protein aggregates. We previously reported that intracellular TGase2 is activated by oxidative stress. To elucidate the functional role of TGase2 activation in cells under the oxidatively stressed condition, we identified the mediator that activates TGase2. In this study, we showed that low levels of oxidative stress trigger the release of TGFβ, which subsequently activates TGase2 through the nuclear translocation of Smad3. Analysis of substrate proteins reveals that TGase2-mediated protein modification results in a decrease of protein solubility and a collapse of intermediate filament network, which leads to aggregation of proteins. We confirm these results using lens tissues from TGase2-deficient mice. Among several antioxidants tried, only N-acetylcysteine effectively inhibits TGFβ-mediated activation of TGase2. These results indicate that TGFβ mediates oxidative stress-induced protein aggregation through activation of TGase2 and suggest that the formation of protein aggregation may not be a passive process of self-assembly of oxidatively damaged proteins but may be an active cellular response to oxidative stress. Therefore, TGFβ-TGase2 pathway may have implications for both the pathogenesis of age-related degenerative diseases and the development of pharmaceutics.—Shin, D.-M., Jeon, J.-H. Kim, C.-W., Cho, S.-Y., Lee, H.-J., Jang, G.-Y., Jeong, E. M., Lee, D.-S., Kang, J.-H., Melino, G., Park, S.-C., Kim, I.-G. TGFβ mediates activation of transglutaminase 2 in response to oxidative stress that leads to protein aggregation.


Key Words: aging • cataract • protein modification


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
TRANSGLUTAMINASE 2 (TGASE2) IS A calcium-dependent enzyme that catalyzes acyl transfer reactions between the {gamma}-carboxamide group of glutamine residues and the {epsilon}-amino group of lysine residues, producing cross-linked, polyaminated, or deamidated proteins (1) . TGase2 has been implicated in various physiological processes, including cell adhesion, extracellular matrix formation, apoptosis, and differentiation (2) . Since TGase2 mediates irreversible modification of substrates that are resistant to proteolytic degradation and, importantly, induces drastic conformational changes of the proteins, it has been suggested that TGase2 may be involved in the formation of insoluble aggregates of proteins (3) .

Aggregation of protein in postmitotic cells is a key feature of age-related degenerative diseases. An increased production of reactive oxygen species (ROS) due to mitochondrial dysfunction in aging causes a variety of oxidative modifications of aggregation-prone proteins such as thiolation, glycation, phosphorylation, and deamidation (4) . It has been widely assumed that oxidatively modified proteins assemble themselves into tangled aggregates when accumulated in sufficient quantity due to the failure of the ubiquitin-proteosome system to degrade the damaged proteins (5) . However, the process that leads to protein aggregation remains to be elucidated.

Recently, we showed that ROS activate intracellular TGase2 in various cell types (6) . In addition to oxidative modification of proteins, ROS induce cellular responses by triggering the activation of a number of signaling pathways such as p53, NF{kappa}B, PI3K/Akt, and Erk/JNK/p38 MAPK signaling pathways (7) . Activation of these pathways induces changes in transcriptional profiles that contribute to cellular senescence and age-related degenerative diseases. Thus, identification of the specific redox-sensitive signaling pathway involved in the activation of TGase2 may help to understand the role of TGase2 in cellular response to oxidative stress.

Toward this end, we chose to study oxidative stress-induced opacification of the lens as a model system. The lens comprises a single layer of epithelial cells that differentiate into new fiber cells, forming lamellae without replacing the older fiber cells (8) . Fiber cells contain a viscous solution of crystallins in an ordered arrangement to maintain lens transparency. Crystallins undergo a variety of post-translational modifications by UV or oxidative stress that may induce a conformational change leading to the misfolding of crystallins (9) . Proteins in lens fiber cells are not replaced after synthesis due to the absence of subcellular organelles and limited proteolysis, accumulating oxidatively modified proteins without degradation (10) . Moreover, aggregation of lens proteins could be easily detected by the development of lens opacity. Thus, the lens is an excellent system to distinguish the aggregates formed by self-assembly of oxidatively modified proteins from the aggregates actively formed by cellular responses to oxidative stress. In this study, we show that TGFβ released by low levels of oxidative stress mediates the activation of TGase2 and that TGase2 plays a critical role in the formation of protein aggregates.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Measurement of intracellular TGase activity
Intracellular TGase2 activity was measured, as described previously, by determining biotinylated pentylamine (BP; Pierce, Rockford, IL, USA) incorporated into cellular proteins (6) . TGase2 activity was expressed as folds of activation compared with the samples without oxidative stress after subtracting the values obtained in the absence of BP, which represent endogenous biotin-conjugated proteins. TGase2 activity was also visualized by probing BP incorporated into cellular proteins with streptavidin-HRP (SA). In vitro TGase2 activity was determined by measuring the incorporation of BP into N,N'-dimethylcasein (Sigma, St. Louis, MO, USA) coated in microplate as described previously (11) . Recombinant TGase2 was purified by a baculovirus expression system (6) .

Cell culture and transfection
Human lens epithelial line (HLE-B3) cells grown in modified Eagle’s medium (MEM) containing 20% fetal bovine serum were maintained in serum-free media for 12 h at 37°C and exposed to media containing H2O2 (Sigma) or TGFβ2 (R&D Systems, Minneapolis, MN, USA). Cystamine (0.25 mM, Sigma) and pan-specific neutralizing antibody for TGFβ (30 µg/ml, R&D Systems) were used to inhibit the TGase2 and TGFβ activities, respectively. Conditioned media (CM) were prepared by centrifugation at 1000 g for 5 min at 4°C and supplemented with protease inhibitor cocktail. To test the effect of antioxidants on TGFβ signaling pathway, HLE-B3 cells were treated with TGFβ2 (5 ng/ml) for 48 h in the presence of vitamin C (1 mM, Sigma), vitamin E (2 mM, Sigma), trolox (0.5 mM, Sigma), quercetin (10 µM, Sigma), and N-acetylcysteine (NAC; 10 mM, Sigma). To inhibit TGFβ2-induced transcription or translation, cells were incubated with actinomycin D (1 µg/ml, Sigma) or cycloheximide (10 µg/ml, Sigma), respectively, for 3 h before treatment of TGFβ2. For UV irradiation, cells (2x106) were exposed to 40, 60, and 80 J/m2 of UVC (UVC-508; Ultra-Lum Inc, Claremont, CA, USA) and further maintained for 24 h. Expression of Smad3, 4 or Smad3DN (12 , 13) was performed by transfection of p6Myc-pCDNA3 vector containing respective cDNA using LipofectAMINE (Invitrogen, Carlsbad, CA, USA).

Measurement of intracellular calcium
Ca2+ levels were measured by fluorimetry, using Fluo-4AM (Molecular Probes, Carlsbad, CA, USA). Approximately 3 x 104 cells were grown overnight in a 96-well microplate. After exposure to H2O2 or TGFβ2, the cells were incubated with 100 µl of assay buffer (Han’s balanced salt solution in 20 mM HEPES, pH 7.4) containing 5 µM Fluo-4AM at 37°C for 30 min and for an additional 30 min at room temperature. The cells were then washed with the assay buffer 4x, and the intensity of fluorescence was measured using a fluorescence microplate reader (Cary Eclipse; Varian, Palo Alto, CA, USA), setting excitation at 488 nm and emission at 516 nm. After reading, the cells were stained with Crystal Violet to normalize the fluorescence value. Intracellular Ca2+ levels were presented as the ratio of value in H2O2-treated cells to that in untreated cells. EGTA (1.5 mM) and BAPTA-AM (20 µM, Molecular Probes) were used to chelate the calcium.

TGFβ assay
p3TP-Luciferase construct (14) and SBE4-luciferase construct (15) were used to monitor the bioactivity of TGFβ in CM. Each construct was cotransfected with vector expressing β-galactosidase. After 24 h of transfection, the cells were treated with H2O2 or TGFβ2. Luciferase activity was assayed using a kit (Promega, Madison, WI, USA) and normalized by β-galactosidase activity. Quantification of TGFβ1 and 2 in CM was performed by using DuoSet kits (R&D Systems).

Western blot analysis
Whole cell extracts were prepared by sonication in buffer (50 mM Tris-Cl, pH 6.8; 6 M urea; 2% SDS; 40 mM dithiothreitol; and protease inhibitor cocktail). After centrifugation (12,000 g, 10 min at 4°C), protein concentration was measured by the BCA method. The cell extracts (30 µg) were separated in 8 or 12% SDS-PAGE. The protein bands were probed with monoclonal antibodies specific for actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), {alpha}-smooth muscle actin (DAKO Corporation, Glostrup, Denmark), fibronectin (Santa Cruz Biotechnology), epitope-Myc (Roche, Basel, Switzerland), TGase2 (16) , vimentin (Santa Cruz Biotechnology), or with polyclonal antibodies specific for {alpha}B-crystallin (Calbiochem, Darmstadt, Germany), βB1-crystallin, and phosphorylated Smad3 (17) , respectively. Monoclonal antibody for TGase2 was generated using recombinant human TGase2 as an antigen (16) . For solubility experiments, the cell extracts were separated into the water-soluble and water-insoluble fractions, as described previously (6) .

Immunocytochemical analysis
Cells were cultured on glass coverslips placed in a 48-well plate for 24 h and exposed to H2O2 or TGFβ2. They were fixed with 4% formaldehyde in PBS for 5 min and permeabilized with 0.1% Triton X-100 in PBS for 5 min at room temperature. The cells were incubated in 3% BSA in PBS at room temperature for 30 min and stained with antibodies specific for Smad3 or vimentin, followed by FITC-conjugated antibody (Jackson Laboratory, Bar Harbor, ME, USA). BP cross-linked with cellular proteins was probed with Texas Red-conjugated streptavidin (Jackson Laboratory). Cells were visualized and photographed with a confocal laser-scanning microscope (LSM510; Carl Zeiss, Thornwood, NY, USA).

Lens organ culture
All animal experiments were approved by Institutional Animal Care and Use Committee of Seoul National University. Preparation and organ culture of lens were performed as described previously (18) . In brief, 20-wk-old male C57BL/6 (TGase2+/+, Charles River Laboratories, Wilmington, MA, USA) and TGase2-deficient (TGase2–/–) mice (19) were anesthetized, and their eyes were enucleated. Lenses were dissected by posterior approach and incubated for 24 h in MEM containing 100 U/ml penicillin, 100 mg/ml streptomycin, and 50 mg/ml gentamicin (pH 7.4, MEM) at 37°C in a humidified CO2 incubator. Lenses were monitored during 24 h in culture, and any lens that had opacity caused by rupture was discarded. Transparent lenses were transferred to a 48-well plate and maintained in MEM in the presence or absence of TGFβ2 (10 ng/ml) with a medium change every 2 days. Lens images were taken at 3, 5, 7, and 10 days following the start of culture under a dark-field microscope. To measure intracellular TGase2 activity, the lenses were incubated with 1 mM BP for 1 h on day 5 or 10 of culture. Rat lenses were prepared as similar to mice lens using 8-wk-old male Sprague-Dawley rats. Rat lenses were cultured in MEM with H2O2 (1 mM) for 5 days, refreshing media every 2 days with fresh H2O2 and cystamine (0.25 mM).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
TGase2 is activated by oxidative stress via two different mechanisms
To identify the mediators that link oxidative stress and the TGase2 activation, we monitored the intracellular TGase2 activity in HLE-B3 cells for 60 h after treatment with various H2O2 concentrations. At a concentration of 200 µM H2O2, the TGase2 activity rapidly rose to a maximum after 12 h. However, at a concentration of 25 µM H2O2 or less, the enzyme activity gradually increased to a maximum after 48 h (Fig. 1 A). The enzyme activity did not correlate with the induction of TGase2 expression (Fig. 1B ), suggesting that two different TGase2 activation mechanisms might be involved, depending on the level of oxidative stress. Because TGase2 is a calcium-dependent enzyme, we measured intracellular calcium concentrations after treatment with 200 or 25 µM of H2O2. Calcium concentration in HLE-B3 cells correlated with the observed time-dependent changes in TGase2 activity after treatment with 200 µM H2O2 but not with 25 µM H2O2 (Fig. 1C ). The TGase2 activity was abrogated in calcium-free media or in the presence of BAPTA-AM (Fig. 1D ), indicating that the elevated intracellular calcium concentration is responsible for the observed rise in TGase2 activity after treatment with 200 µM H2O2.


Figure 1
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  		Figure 1. Oxidative stress activates TGase2 by increase of intracellular calcium concentration. A) TGase2 activity of HLE-B3 cells was monitored for 60 h following exposure to H2O2 at various concentrations. B) Expression of TGase2 in HLE-B3 cells after exposure to H2O2 was examined by Western blot analysis. Actin was used as a loading control. C) Change in [Ca2+]i in response to H2O2. D) Intracellular TGase activity was measured in HLE-B3 cells after exposure to 200 µM H2O2 for 12 h in the presence or absence of either EGTA (1.5 mM) or BAPTA-AM (20 µM).

The delay in TGase2 activation at a concentration of 25 µM H2O2 indicates an involvement of additional steps in the activation of TGase2. Oxidative stress triggers either a release or activation of some cytokines, such as IL-8, TNF{alpha}, and TGFβ (20) . Particularly, TGFβ is reported to induce the formation of protein aggregates in transgenic mice and also in lens organ culture system (21 , 22) . We investigated whether TGFβ is involved in the delayed TGase2 activation. TGFβ bioassay was performed using HLE-B3 cells that are transfected with p3TP-luciferase reporter, which contains three tetradencanoyl phorbol acetate response elements (TRE) and the TGFβ responsive element of the human plasminogen activator inhibitor-1 (PAI-1) promoter (14) . The time course of luciferase activity induced by CM (prepared from the culture treated with H2O2) resembled that of the delayed activation of TGase2 by oxidative stress (Fig. 2 A). The CM, prepared with the cells treated with 25 µM H2O2, produced a maximum luciferase activity associated with p3TP-luciferase reporter after 48 h.


Figure 2
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  		Figure 2. Oxidative stress induces a release of TGFβ2 that activates TGase2. A) Bioassay for TGFβ in CM prepared from HLE-B3 cells following exposure to various H2O2 concentrations. Luciferase activity was measured in HLE-B3 cells transfected with p3TP-luciferase reporter construct. Cells were exposed to CM for 24 h. B) Luciferase activity in HLE-B3 cells following exposure to CM in the presence of TGFβ-neutralizing antibody. CM was prepared from cells treated with 25 µM H2O2. TGFβAb, TGFβ-neutralizing antibody (30 µg/ml). C) Western blot analysis for fibronectin (FN), {alpha}-smooth muscle actin ({alpha}-SMA), and TGase2 with HLE-B3 cells incubated with CM for 24 h. D, E) Measurement of active TGFβ1 (D) and TGFβ2 (E)in CM by ELISA. Data are shown as means ± SD; n = 3. F) Effect of TGFβ-neutralizing antibody on TGase activity. G) Effect of TGFβ-neutralizing antibody on TGase activity and expression of FN induced by UVC irradiation. TGase2 activity was measured after 24 h of UVC irradiation in the absence or presence of TGFβ-neutralizing antibody.

We examined the effect of TGFβ-neutralizing antibody on luciferase and TGFβ-induced genes. Addition of the antibody suppressed the rise of luciferase activity as well as CM-induced expression of {alpha}-smooth muscle actin ({alpha}-SMA) and fibronectin (Fig. 2B, C ). Measurements of TGFβ concentration in the CM revealed that the level of TGFβ2, but not that of TGFβ1, directly correlates with TGase2 activation (Fig. 2D, E ). Furthermore, we confirmed that TGFβ-neutralizing antibody suppressed the rise of TGase2 activity caused by H2O2 treatment at concentration of 25 µM or lower (Fig. 2F ), indicating that low levels of H2O2 trigger a release of TGFβ2, which subsequently activates TGase2. We next tested whether TGFβ signaling pathway is also involved in the TGase2 activation in response to UV irradiation. When HLE-B3 cells were exposed to subcytotoxic doses of UVC irradiation, TGFβ-neutralizing antibody did not suppress the increase of TGase2 activity by UV irradiation (Fig. 2G ). These results suggest that TGFβ-mediated TGase2 activation is dependent on the type of oxidative stress.

TGFβ activates TGase2 in a Smad3-dependent manner
TGFβ binds to its receptors, which initiate intracellular signaling by phosphorylation of receptor-regulated Smad (R-Smad), such as Smad2 and Smad3 (23) . The phosphorylated R-Smad forms a complex with Smad4, which translocates to nucleus where it regulates the transcription of TGFβ-dependent gene targets. We investigated whether Smad3 mediates TGFβ-mediated TGase2 activation. In HLE-B3 cells, TGFβ2 increased TGase activity in a dose- and time-dependent manner (Fig. 3 A, B). Oxidative stress or TGFβ2 induced the phosphorylation of Smad3 (Fig. 3C ). Immunocytochemical staining showed that TGase activity was associated with nuclear translocation of Smad3 and that TGFβ-neutralizing antibody abrogated the nuclear translocation of Smad3 induced by either H2O2 or TGFβ2 (Fig. 3D ).


Figure 3
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  		Figure 3. Smad3 signaling pathway is required for TGase2 activation. A) A dose-dependent activation of TGase2 by TGFβ2 in HLE-B3 cells. TGase activity was measured after 48 h of TGFβ2 treatment. B) A time-dependent activation of TGase2 by TGFβ2 (5 ng/ml). Cells were incubated with BP (1 mM) for 1 h to determine the intracellular TGase activity. C) Phosphorylation of Smad3 in response to H2O2 or TGFβ2 (1 ng/ml). Arrow, phospho-Smad3; asterisk, nonspecific bands. D) Nuclear translocation of Smad3 (green) associated with TGase activity (red) in the cells treated with either H2O2 (25 µM) or TGFβ2 (5 ng/ml) for 48 h. Streptavidin-HRP was used to detect the BP incorporated into proteins. TGFβAb, TGFβ-neutralizing antibody (30 µg/ml); Cyst, cystamine (0.25 mM). E) Effect of overexpression of Smad3 and Smad4 on TGase2 activity. After 48 h with Smads transfection, TGase activity was measured. F) Effect of dominant-negative Smad3 (Smad3DN) on TGase2 activity. After 24 h of Smad3DN transfection, the cells were treated with either H2O2 (25 µM) or TGFβ2 (5 ng/ml). TGase activity was measured after 48 h. G) Effect of pretreatment with actinomycin D (ActD, 1 µg/ml) or cycloheximide (CHX, 10 µg/ml) for 3 h on TGase activity and expression of FN induced by TGFβ2 (5 ng/ml). TGase activity and expression of FN were measured after 24 h of TGFβ2 treatment.

To confirm the involvement of Smad3 signaling pathway in TGase2 activation, Smad3 was overexpressed alone or with Smad4. Overexpression of Smad3 alone did not affect TGase2 activity. In contrast, cotransfection of Smad3 and Smad4 raised the intracellular TGase activity ~3.5-fold (Fig. 3E ). Particularly, the overexpression of dominant-negative Smad3, in which phosphorylation site was deleted (13) , suppressed the rise of TGase2 activity induced by either H2O2 or TGFβ2 treatment (Fig. 3F ), indicating that TGFβ2 or a low concentration of H2O2 activated TGase2 through Smad3 signaling pathway. In addition, when HLE-B3 cells were treated with actinomycin D or cycloheximide, TGFβ2-mediated TGase2 activation was abrogated (Fig. 3G ). These results indicate that de novo expression of Smad3-dependent genes is required for activating TGase2 without increasing protein level.

We next assessed the effect of antioxidants on TGFβ2-mediated activation of TGase2. The presence of vitamin C, vitamin E, trolox, or quercentin did not influence the TGase2 activation. In contrast, NAC, under the same conditions, effectively suppressed the activation of TGase2 induced by TGFβ2 (Fig. 4 A). NAC did not inhibit the activity of purified TGase2 but suppressed the TGFβ-induced expression of fibronectin (Fig. 4A, B ). This observation suggests that NAC suppresses TGase2 activation by inhibiting TGFβ signaling pathway. To test this possibility, we examined the effect of NAC on luciferase activity in HLE-B3 cells, which had been transfected with a reporter construct that contained four tandem Smad binding element sites (SBE4-Luc) (15) . The elevated luciferase activity, induced by either H2O2 or TGFβ2 treatment, was completely abolished by NAC (Fig. 4C ). Interestingly, cystamine, a low-molecular-weight thiol compound used as a TGase inhibitor (11) , suppressed the expression of fibronectin induced by TGFβ2 (Fig. 4A ). We also found that the increase of luciferase activity of both p3TP-luciferase and SBE4-luciferase reporters induced by TGFβ2 was partially abolished by cystamine (Fig. 4D ). Moreover, the nuclear translocation of Smad3 induced by either H2O2 or TGFβ2 treatment was partially inhibited by cystamine (Fig. 3D ). These results suggest that NAC and cystamine share the common mechanism involving reaction with oxidized proteins to regenerate the glutathione by mixed disulfide exchange (24) . Furthermore, the cross-linking of fibronectin by TGase2 was partially inhibited by cystamine and completely inhibited by NAC (Fig. 4E ). This result indicates that cystamine or NAC has a different target molecule in the TGFβ signaling pathway.


Figure 4
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  		Figure 4. NAC or cystamine inhibits TGFβ2 signaling in HLE-B3 cells. A) Western blot analysis of intracellular TGase2 activity, FN, and TGase2 in HLE-B3 cells treated with TGFβ2 (5 ng/ml) for 48 h in the presence of vitamin C (VitC, 1 mM), vitamin E (VitE, 2 mM), trolox (TX, 0.5 mM), quercetin (QC, 10 µM), cystamine (Cyst, 0.25 mM), and NAC (10 mM). B) Effect of NAC on the activity of recombinant TGase2 purified by baculovirus expression system. C) Effect of NAC (10 mM) on luciferase activity in the cells treated with either H2O2 or TGFβ2 (1 ng/ml) for 24 h. D) Luciferase activity was measured in HLE-B3 cells transfected with p3TP-luciferase or SBE4-luciferase construct. Cells were treated with TGFβ2 (1 ng/ml) for 24 h in the presence or absence of Cyst (0.25 mM). E) Effect of Cyst (0.25 mM) or NAC (10 mM) on TGase activity and expression of FN induced by TGFβ2 (5 ng/ml). SA, streptavidin-HRP.

TGase2-mediated modification is critical for protein aggregation
We next tested whether TGase2-mediated modifications occurring under oxidative stress play a causal role in protein aggregation. To examine whether proteins could be modified by TGase2 under oxidative stress condition, HLE-B3 cells were treated with H2O2 or TGFβ2 and then incubated with BP. Proteins cross-linked with BP by TGase2 were separated using streptavidin and subjected to Western blot analysis. Figure 5 A shows that {alpha}B-crystallin, βB1-crystallin, vimentin, and fibronectin were cross-linked with BP in cells treated with H2O2 or TGFβ2 and that BP incorporation was inhibited by cystamine or TGFβ-neutralizing antibody. We then examined the effect of TGase2 activation on the solubility of substrate proteins. HLE-B3 cells were treated with H2O2 or TGFβ2 and separated into soluble and insoluble fractions. {alpha}B- and βB1-crystallins were found in the insoluble fraction following treatment with H2O2 or TGFβ2. The insoluble proteins, however, disappeared when either cystamine or TGFβ-neutralizing antibody was added to the culture media. In contrast, the amount of insoluble vimentin did not change even after a treatment with H2O2 or TGFβ2 (Fig. 5B ). Instead, vimentin was found to be cross-linked under the same experimental conditions. The cross-linked vimentin disappeared when either cystamine or TGFβ-neutralizing antibody was added (Fig. 5C ). Immunocytochemical staining for vimentin revealed that the cross-linking of vimentin induced the collapse of intermediate filament network (Fig. 5D ). These findings demonstrate that TGase2-mediated modifications induce the aggregation of proteins by lowering their solubility and increasing the cross-link between the proteins.


Figure 5
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  		Figure 5. TGase2-mediated modification results in the aggregation of lens proteins. A) Lens proteins as substrates for TGase2 activated by H2O2 (25 µM) or TGFβ2 (5 ng/ml) for 48 h. Lens proteins incorporated with BP were separated using streptavidin and subjected to Western blot analysis. Cryab, {alpha}B-crystallin; Crybb1, βB1-crystallin. B) Solubility of modified crystallins assessed by separating the cell extracts into soluble (Sol) and insoluble (InS) fractions. TGFβAb, TGFβ-neutralizing antibody (30 µg/ml); Cyst, cystamine (0.25 mM). C) Cross-linked vimentin in the whole cell extracts. D) Immunocytochemical staining of vimentin after exposure to TGFβ2 (5 ng/ml) for 48 h. DAPI-stained DNA was used to identify nuclei.

To further substantiate the causal role of TGFβ-mediated TGase2 activation in protein aggregation, in vivo experiments were performed with a mouse model of cataractogenesis, using a lens organ culture system (18) . Cortical opacification developed in the lens of wild-type mice on day 5 following the treatment with TGFβ2, progressing to complete opacity on day 10. The development of opacity could be partially delayed by the administration of cystamine. In contrast, TGFβ2 did not induce opacity in the lens of TGase2-deficient mice up to 10 days (Fig. 6 A). TGase activity in the mouse lens was measured at day 5, and it showed a close correlation with the opacity of the lens (Fig. 6B ). We also exposed rat lens to 1 mM of H2O2 and confirmed that cystamine prevented the development of opacity in the lens (Fig. 6C ). Taken together, these results provide support to the notion of novel role for TGase2 as a key enzyme in mediating oxidative stress-induced protein aggregation.


Figure 6
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  		Figure 6. Activation of TGase2 is critical in oxidative stress-induced protein aggregation. A) Images of lenses taken from TGase2+/+ and TGase2–/– mice cultured in MEM with TGFβ2 (10 ng/ml) for 10 days. Medium was changed every 2 days with fresh TGFβ2, cystamine (0.25 mM) or TGFβ-neutralizing antibody (30 µg/ml). B) TGase activity in the lens at day 5. Lenses were cultured with BP (1 mM) for 1 h prior to assay. C) Images of lenses from Sprague-Dawley rats cultured in MEM with H2O2 (1 mM) for 5 days, changing medium every 2 days with fresh H2O2 and cystamine (0.25 mM).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
TGases are a family of enzymes that catalyze the formation of {epsilon}-({gamma}-glutamyl)lysine isopeptide bonds between a number of substrate proteins. This cross-linking reaction plays a key role in the formation of barrier structures in skin (TGase1, TGase3, and TGase5), fibrin aggregation in blood clots (coagulation factor XIIIa), and postcoital plug of seminal fluid (TGase4) through activation of proenzymes by proteolysis (25 26 27) . TGase2 is unique among TGase family for its ubiquitous expression and widespread subcellular localization (2) . Because of low substrate specificity, it has been thought that activation of TGase2 and resulting cross-linking of intracellular proteins may negatively affect cellular function. However, the functional role of intracellular TGase2 activity remains unclear. In this study, we identified TGFβ as a mediator that links oxidative stress and TGase2 activation, suggesting a new role of TGFβ in protein aggregation. In addition, we have shown that TGase2 plays an essential role in the formation of protein aggregates by modifications of substrate proteins that decrease the solubility of proteins and disrupt their cytoskeletal structures (Fig. 7 ).


Figure 7
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  		Figure 7. A plausible mechanism for protein aggregation based on the present results.

Our results would assist understanding the pathogenesis of diseases associated with oxidative stress and protein aggregation. Cataract is a common, age-related disorder caused by aggregation of lens proteins. Epidemiological studies on risk factors suggest a causal role of oxidative stress in the pathogenesis (28 , 29) . However, the molecular mechanisms underlying this process are not yet elucidated. With aging, crystallins undergo a variety of post-translational modifications by UV or oxidative stress due to mitochondrial dysfunction. However, since modified proteins are found in normal aged lens as well as cataractous lens (30) , a causal relationship between any specific type of modification of lens proteins and cataractogenesis has not been definitely established. Our experiments using lens from TGase2 knockout mouse and inhibitors demonstrated that modifications of lens proteins catalyzed by TGase2 are critical for cataractogenesis. Thus, an accumulation of modified proteins by TGase2 to a certain threshold level may be required for cataract formation. In addition, the finding that NAC and cystamine can effectively inhibit TGase2 activation provides a rational basis for the development of new compounds that may prevent or delay cataract progression.

In a normal culture system, intracellular TGase2 activity was not detectable in lens epithelial cells despite a positive expression of TGase2, indicating that TGase2 is tightly regulated in intracellular environment. Although we identified TGFβ2 as a signaling pathway that mediates oxidative stress-induced activation of TGase2, two important issues remain unresolved. The first relates to identifying the molecules responsible for activating TGFβ. We showed that a low level of oxidative stress increases the concentration of active TGFβ2. An increase in TGFβ1 mRNA/bioactivity has been observed in mammary glands after irradiation (31) , in fibroblasts in which premature senescence was induced by exposure to UVB (32) , and in kidney mesangial cells treated with β-hydroxybutyrate (33) . However, the mechanism for activation of latent TGFβ by oxidative stress is unknown. Although it has been reported that recombinant latent TGFβ1 is activated by ROS through direct oxidation of cysteine or methione (34) , further study is needed to identify the molecules responsible for activating TGFβ. The second issue relates to finding that activation of TGase2 is dependent on the nuclear translocation of Smad3-Smad4 complex and that TGFβ2-mediated TGase2 activation is completely abrogated by pretreatment with actinomycin D or cycloheximide. Molecules induced by Smad3 remain to be identified, and this is important to understand the mechanism for TGFβ-mediated TGase2 activation.

Calcium is known to activate transamidation activity of TGase2 that is inhibited by GTP binding (1) . Our results demonstrated that oxidative stress raises the level of intracellular calcium that is responsible for the activation of TGase2. Activation of TGase2 by oxidative stress dose not occur in calcium-free media, indicating that the source of calcium might be extracellular and that the elevated influx of calcium is probably due to the activation of TRPM2 channel, a transient receptor potential cation channel that is sensitive to oxidative stress (35) . Therefore, perturbation of calcium homeostasis could explain the increased susceptibility of aged cells to oxidative stress-induced protein aggregation. Furthermore, a decreased concentration of GTP due to impaired metabolic function in aged cells may also contribute to aberrant activation of TGase2.

TGase2 is involved in the formation of stress fiber and focal adhesion complex through modification of RhoA (36) . Polyamination or deamidation of glutamine 63 of RhoA by TGase2 causes a decrease in its GTPase activity. As a result, RhoA becomes constitutively active and binds Rho-associated kinase 2, thereby inducing polymerization of actin. Interestingly, our study reveals another mechanism by which TGase2 modulates cytoskeletal organization, namely, via the disruption of intermediate filament network through direct cross-linking of vimentin. Therefore, TGase2 may be responsible for the changes in cell morphology observed in oxidative stress-induced premature senescence.

Our identification of TGFβ as a mediator that activates TGase2 also has implications for the role of TGase2 in fibrotic diseases. TGFβ is a multifunctional growth factor involved in the modulation of cell proliferation, cell death, and tissue repair (37) . TGFβ enhances various extracellular matrix proteins, resulting in tissue fibrosis. Our results revealed that TGase2 is a downstream effector of TGFβ. Accordingly, TGase2 activation may contribute to the pathogenesis of various fibrotic diseases in which the concentration of TGFβ increases in response to oxidative stress. Such diseases include diabetic retinopathy, hepatic fibrosis, bleomycin-induced pulmonary fibrosis, and proliferative vitreoretinopathy (38 39 40 41) . Further studies are needed using a specific animal model to determine the effects of TGase2 inhibition on disease progression.


   ACKNOWLEDGMENTS
 
We thank Dr. Y. D. Kim and Dr. J. S. Ram for critical comments, Y. J. Kim for helping with lens organ culture, Dr. Joan Massague (Memorial Sloan-Kettering Cancer Center, New York, NY, USA) for kindly providing the p3TP-Lux construct, Dr. Masahiro Kawabata (Japanese Foundation for Cancer Research, Tokyo, Japan) for cDNA constructs of Smad3 and 4, Dr. Seong-Jin Kim (U.S. National Institutes of Health, Bethesda, MD, USA) for SBE4-luciferase construct, Dr. Edward B. Leof (Mayo Clinic College of Medicine, Rochester, MN, USA) for polyclonal antibody specific for phosphorylated Smad3, and Dr. Joseph Horwitz (University of California, Los Angeles, School of Medicine, Los Angeles, CA, USA) for polyclonal antibody specific for βB1-crystallin. This work was supported by grants from the Korea Science and Engineering Foundation (R11-2002-097-07002-0 and R01-2005-000-10364-0) through the Aging and Apoptosis Research Center. G.Y.J. and E.M.J. were supported by the graduate program of BK21 from the Ministry of Education and Human Resources Development.

Received for publication August 28, 2007. Accepted for publication February 21, 2008.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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S. E. Iismaa, B. M. Mearns, L. Lorand, and R. M. Graham
Transglutaminases and Disease: Lessons From Genetically Engineered Mouse Models and Inherited Disorders
Physiol Rev, July 1, 2009; 89(3): 991 - 1023.
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