Upregulation of TGF-β–Induced Tissue Transglutaminase Expression by PI3K-Akt Pathway Activation in Human Subconjunctival Fibroblasts

Abstract

purpose. Excessive scarring in subconjunctival tissues after filtering surgery seems to be characterized by aberrant extracellular matrix (ECM) production, and tissue transglutaminase (tTgase) plays an important role in this process. In the present study, the effects of transforming growth factor (TGF)-β2 on the expression of tTgase, its activity in subconjunctival fibroblasts and whether the effects of TGF-β are mediated by prosurvival signaling pathways were examined.

methods. Primary subconjunctival fibroblasts treated with TGF-β2 were examined for the expression of tTgase with Western blot analysis. The modulation of extracellular tTgase activity by TGF-β2 was measured by both the formation of fibronectin polymers and the ECM protein incorporation of fluorescein cadaverine. The expression of tTgase was analyzed by immunofluorescence staining and Western blot analysis of subconjunctival fibroblasts that were transiently transfected with an Akt dominant negative mutant gene or were treated with an Akt inhibitor or tTgase siRNA.

results. Treatment of subconjunctival fibroblasts with TGF-β2 caused an increase in activation and expression of tTgase. The effects of TGF-β stimulation of subconjunctival fibroblasts were twofold, causing both rapid activation of the ERK pathway within minutes of treatment and a more delayed activation of the phosphatidylinositol3-kinase-protein kinase B (PKB)/Akt pathway; however, only Akt activation was necessary for TGF-β-induced tTgase expression. Transient transfection of subconjunctival fibroblasts with an Akt dominant negative mutant gene, or treatment with an Akt inhibitor (but not with an ERK inhibitor) or tTgase siRNA led to decreased activation and expression of tTgase.

conclusions. TGF-β2 activated the PI3K-Akt pathway, and this activation was essential for the expression and activity of tTgase in subconjunctival fibroblasts. The results indicate a novel biological function of the PI3K-Akt pathway in subconjunctival fibroblasts. Elevated expression and activity of tTgase may play an important role in the pathogenesis of diseases related to wound healing and fibrogenic reactions in subconjunctival fibroblasts.

Conjunctival and scleral scarring remain as limiting factors in the success of filtration surgery, and scarrings in the sclerostomy site or bleb connective tissue are formed by excessive proliferation and aberrant extracellular matrix (ECM) production beneath the conjunctiva. Thus, agents inhibiting fibroblast proliferation such as 5-FU or mitomycin C play an important role in increasing the success rate of filtering surgery. ^{1 2} Reports have shown that transforming growth factor (TGF)-β is a key modulator in this disease. ^{3 4} TGF-β regulates cellular proliferation, differentiation, and other functions in many cell types, ^{3 4} and increased expression of TGF-β associated with subconjunctival fibrosis has been shown to occur in bleb scarring. ^{1 2} Much information has recently been obtained concerning the signal transduction pathways by which the cellular actions of TGF-β are controlled and how the actions of this protein are enhanced or suppressed by various factors. ⁴ TGF-β, a universal inhibitor of cell proliferation, stimulates the cellular proliferation of fibroblasts and has direct and indirect mitogenic effects in subconjunctival fibroblasts through an autocrine response. ⁵  

Tissue transglutaminase (tTgase) is a ubiquitously expressed enzyme that modifies proteins by cross-linking or polyamidation. ^{6 7} Tgases are a Ca²⁺-dependent family of enzymes that establish ε-(γ-glutamyl) lysine cross-linkages and aid in the incorporation of polyamines and histamine into proteins via covalent bonding, ^{6 7} and this catalytic action of Tgases results in the formation of an isopeptide bond that is highly resistant to both proteolysis and denaturants. The Tgase family is composed of several members, including plasma factor XIII, Tgase 1 (keratinocyte Tgase), tTgase (tissue Tgase), Tgase 3 (epidermal Tgase), and Tgase 4 (prostate Tgase). Among the Tgase family members, tTgase has been the most widely identified, occurring in a variety of cell types, ⁸ and has been implicated in diverse physiological functions such as cell differentiation, ⁹ wound healing, ¹⁰ and apoptosis. ¹¹ Further, tTgase can be involved in the cross-linking of both intracellular and extracellular proteins. For example, tTgase appears to be involved in the cross-linking of intracellular proteins in cells undergoing apoptotic cell death. ¹¹ tTgase is also secreted onto the surface of the cells and plays a key role in the cross-linking of extracellular matrix proteins. ^{12 13 14}  

Despite these data, the molecular mechanism by which tTgase is activated has not yet been elucidated. We speculated that tTgase may act as an important pathogenic modulator in the matrix-remodeling processes in subconjunctival fibroblasts via TGF-β. To test this hypothesis, we first investigated the expression of tTgase in cultured human subconjunctival fibroblasts and determined whether TGF-β increases tTgase expression and its activation. We subsequently investigated whether TGF-β used a prosurvival signaling pathway to mediate tTgase expression and its activation in subconjunctival fibroblasts. 

Materials and Methods

Cell Culture and Treatment

In compliance with the provisions of the Declaration of Helsinki, human subconjunctival fibroblasts were obtained from excised Tenon’s capsule specimens during strabismus surgery. Written informed consent was obtained before operative excision. Institutional human experimentation committee approval was also granted. Briefly, 5 × 5-mm sections of Tenon’s capsule were collected, minced, and placed in a 35-mm culture dish containing Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum, 50 μg/mL penicillin, and 50 μg/mL streptomycin (DMEM-10). Cells were allowed to migrate from the explant tissue and were then incubated at 37°C in a 5% CO₂ environment. When a confluent monolayer of the primary culture stage was obtained, the cells were incubated with 0.05% trypsin and 5 mM EDTA at 37°C for 5 minutes and transferred to a 100-mm culture dish containing DMEM-10. Cells that maintained proliferative potential and fibroblast-like elongated morphology between the third and fifth passages were used for this study. Cultures were allowed to reach 75% to 80% confluence, and were supplemented with 10 ng/mL of TGF-β2 or TGF-β1 (R&D System, Minneapolis, MN) in serum-free medium. 

Western Blot Analysis

Total cell lysates were obtained from cultured subconjunctival fibroblasts by using lysis buffer (25 mM HEPES [pH 7.5], 0.3 M NaCl, 1.5 mM MgCl₂, 0.2 mM EDTA, 0.05% Triton X-100, 0.5 mM dithiothreitol [DTT], and 0.4 mM phenylmethylsulfonyl fluoride [PMSF]; Sigma-Aldrich, St. Louis, MO), 2 μg/mL leupeptin (Sigma-Aldrich), and 2 μg/mL aprotinin (Sigma-Aldrich). After centrifugation for 10 minutes at 12,000g, the supernatant proteins (5 μg/lane) were subjected to SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Hybond; GE Healthcare, Piscataway, NJ). Membranes were blocked with TBST (20 mM Tris, 137 mM NaCl [pH 7.4], and 0.02% Tween 20) containing 5% nonfat dry milk, incubated with various primary antibodies diluted in TBST for 24 hours at 4°C, and washed three times with TBST. 

Detection of primary antibodies was achieved by incubating membranes with horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibody diluted 1:5000 in TBST for 1 hour, which was followed by three washes with TGST. Immunoreactive proteins were visualized using chemiluminescence detection reagents (ECL; GE Healthcare) on autoradiograph films. Blots were stripped and reprobed according to the manufacturer’s instructions (GE Healthcare). The anti-tTgase antibody was from NeoMarkers (Fremont, CA); anti-fibronectin antibody, anti phospho-AKT-1, anti-AKT-1, antiphospho-ERK1/2, anti-ERK1/2, antiphospho-JNK, anti-JNK, antiphospho-p38, and anti-p38 MAPK antibody were all obtained from Sigma-Aldrich. Anti-phospho-PI3K and anti-PI3K antibody were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). 

Detection of Extracellular tTgase Activity

An in situ TGase2 activity assay was performed as previously described. ¹⁵ Briefly, the cells were seeded into four-well glass chamber slides (Nunc, Naperville, IL). On reaching 60% to 65% confluence, the cells were washed and incubated in the presence or absence of 10 ng/mL TGF-β2 or -β1 for 72 hours, followed by an incubation with 0.5 mM fluorescein cadaverine (Invitrogen-Molecular Probes, Eugene, OR) for 15 hours. After incubation, the cells were washed, fixed in methanol at −20°C, and mounted. In double-staining experiments (after staining with fluorescein cadaverine and fixing) cells were blocked with 3% BSA in PBS, incubated with rabbit anti-fibronectin antibody (Sigma-Aldrich) diluted 1: 50 in blocking buffer for 15 hours at 4°C, and then incubated with rhodamine-conjugated (TRITC) anti-rabbit IgG (Sigma-Aldrich) diluted 1:50 in blocking buffer for 2 hours at room temperature. Rhodamine-conjugated (TRITC) anti-rabbit IgG alone did not show any reactivity with the cells (data not shown). Cells were viewed using a confocal laser microscope (CLSM; Leica, Heidelberg, Germany) equipped with an argon krypton laser set at 488 and 560 nm for fluorescein and rhodamine excitation, respectively. 

Western Blot Analysis of tTgase and Fibronectin in Cells Transiently Transfected with Akt1(K179M) Gene, a Dominant Negative Mutant Gene

For transient transfection of subconjunctival fibroblasts with a dominant negative mutant (Akt1(K179M) gene (Upstate Biotechnology, Lake Placid, NY), cells were transfected with either pUSEamp-Akt1(K179M) or pUSEamp as a control, using transfection reagents (FuGENE 6; Roche Diagnostics, Indianapolis, IN) according to the manufacturer’s instructions. Briefly, 1 × 10⁵ cells per well were plated in a six-well culture dish and transfected with 1 μg of pUSEamp-Akt1(K179M) or pUSEamp. After 72 hours, cells were collected for Western blot analysis. 

Quantitative Assay for Cell Surface tTgase Activity

Tgase activity associated with the extracellular surface was measured by fibronectin incorporation of biotinylated cadaverine. ¹⁶ For this assay, 2 × 10⁵ cells/mL were plated into 96-well plates precoated with plasma fibronectin in 100 μL complete DMEM without serum but containing 0.1 mM biotinylated cadaverine. As a negative control, 96-well plates coated with fibronectin were incubated with 100 μL serum-free DMEM containing 0.1 mM biotinylated cadaverine in the absence of cells. Subconjunctival fibroblasts were incubated for 1 hour at 37°C and then washed twice with PBS (pH 7.4), containing 3 mM EDTA, to stop the reaction. A detergent solution (100 μL) consisting of 0.1% (wt/vol) deoxycholate in PBS (pH 7.4) and 3 mM EDTA was then added to each well followed by incubation with gentle shaking for 20 minutes. The supernatant was discarded and the remaining fibronectin layer washed three times with Tris-HCl (pH 7.4). Cells were blocked with 3% (wt/vol) BSA in Tris-HCl buffer for 30 minutes at 37°C and washed with buffer. The incorporated biotinylated cadaverine was revealed using a 1:5000 dilution of peroxidase conjugate (Extravidin; Sigma-Aldrich) which was incubated for 1 hour at 37°C with TMB as a substrate. Color development was stopped by adding 50 μL stop solution to each well. The resultant color was then read with an ELISA plate reader at 450 nm. 

RNA Interference Assay

To block the function of tTgase, we used small interfering (si)RNA molecules targeted at tTgase mRNA. A 21-nucleotide (nt) siRNA) sequence was derived from a rat tissue type Tgase II mRNA sequence (GenBank accession no. GI: 42476286; http://www.ncbi.nlm.nih.gov/Genbank; provided in the public domain by the National Center for Biotechnology Information, Bethesda, MD) and obtained from Ambion, Inc. (Austin, TX): siRNA against tTgase (sense, 5′-GGGUUACCGGAAUAUCAUCTT-3′; antisense, 5′-GAUGAUAUUCCGGUAACCCTT-3′). Subconjunctival fibroblasts were transfected with si-tTgase duplexes (siPORT NeoFX; Ambion Inc.). Briefly, the RNA duplex (10 nM final concentration) was incubated in serum-free DMEM containing 5 μL of the transfection reagent for 10 minutes. The complex was then added to the empty wells of a culture plate where the cell suspension containing the transfection complexes (5 × 10⁵ cells/mL) was overlaid onto the culture plate wells. Transfected cells were incubated in normal cell culture conditions until ready for analysis. 

Results

Enhanced Expression of tTgase in Cultured Subconjunctival Fibroblasts Treated with TGF-β2

We first examined whether TGF-β2 was capable of increasing the expression of tTgase in subconjunctival fibroblasts. As shown in Figure 1a , the expression of tTgase protein was substantially increased in a time-dependent manner after the cells were treated with 10 ng/mL TGF-β2, and this increased level of expression was maintained for 2 days. Also, expression of tTgase protein was increased in a dose-dependent manner (Fig. 1b)2 days after treatment with different concentrations of TGF-β2. The expression of tTgase mRNA was also increased in both time- and dose-dependent patterns in the cells treated with TGF-β2 (data not shown). Similar effects were observed after treatment of the cells with TGF-β1 (data not shown). 

Induction of Erk1/2 and Akt Phosphorylation by TGF-β2

To determine whether TGF-β2 stimulates the phosphorylation of Erk1/2, p38 MAPK, JNK and Akt, we stimulated subconjunctival fibroblasts with TGF-β2 and assayed for Erk1/2, p38 MAPK, JNK, and Akt in the cells by immunoblot analyses conducted at the indicated times. Abundance of phosphorylated Erk increased at 5 minutes, was maximized at 30 minutes, and returned to nearly basal levels at 60 minutes (Fig. 2) . In contrast, the level of phosphorylated Akt did not increase at any early time point. Phosphorylation of Akt increased in a delayed manner at 4 hours and was at its maximum at 24 hours. Phosphorylated PI3K followed the same temporal pattern of expression as phosphorylated Akt. JNK and p38 MAPK were not activated in these cells. The bottom panel of Figure 2shows that nonphosphorylated Akt and PI3K protein levels remained constant in each sample. Even though there was some variation in the expression of total Erk1/2 throughout the time course, we think this difference of total Erk1/2 is not significant with respect to the phosphorylation of Erk1/2. Together, these data indicate that the TGF-β2 induced phosphorylation of Erk1/2 and Akt, and the PI3K phosphorylation was most likely a result of posttranslational modifications rather than a change in protein expression patterns (Fig. 2) . 

Effect of LY294002 on Expression of tTgase and its Extracellular Activity Induced by TGF-β2

To determine whether the TGF-β2-stimulated expression and extracellular activity of tTgase correlate with the phosphorylation of Erk1/2 or Akt, cells treated with or without TGF-β2 were cultured in the presence of LY294002 (an Akt phosphorylation inhibitor) or U0126 (an Erk1/2 phosphorylation inhibitor) and examined by Western blot analysis. LY294002 inhibited phosphorylation of PI3K, which indirectly inhibited Akt. As shown in Figure 3a , decreased amounts of tTgase were detected from the lysates of cells treated with TGF-β2 and LY294002 (lane 3) relative to cells treated with TGF-β2 (lane 2). In contrast, however, there was little if any loss of expression of tTgase in lysates of cells treated with TGF-β2 and U0126 (lane 4) relative to cells treated with TGF-β2 (lane 2). These results indicate that the decreased expression of tTgase was coincident with the inhibition of Akt phosphorylation but not of Erk1/2 phosphorylation in subconjunctival fibroblasts. 

In previous reports, tTgase activity was visualized in situ by incorporation of the specific substrate fluorescein cadaverine. ¹⁵ Fluorescein cadaverine is believed to act as a competitive primary amine to the peptide-bound ε-amino lysine group and thus becomes incorporated into endogenous γ-glutamyl residues if active tTgase is present. As shown in Figure 3b , cells exhibited a strong pattern of punctuate patches of fluorescein cadaverine staining on the surface of the cells after treatment with TGF-β2, indicating enhanced tTgase activity (Fig. 3bD) ; a much weaker staining pattern was observed in the nontreated control cells (Fig. 3bA) . Antibodies for fibronectin revealed very few fibronectin fibrils in the nontreated control cells (Fig. 3bB) , whereas abundant fibrils were observed on the surface of the cells treated with TGF-β2 (Fig. 3bE) . Superimposed images depicted the increased colocalization of fluorescein cadaverine and fibronectin in the cells treated with TGF-β2 (Fig. 3bF)relative to the nontreated control cells (Fig. 3bC) . Collectively, these results indicate that tTgase may be directly involved in the cross-linking of fibronectin. The addition of LY294002 to the cells treated with TGF-β2 led to a considerable reduction in incorporation of fluorescein cadaverine (Fig. 3bG) , fibril formation of fibronectin (Fig. 3bH) , and their colocalization (Fig. 3bI) . The addition of U0126 to the cells treated with TGF-β2 led to little if any reduction of fluorescein cadaverine incorporation (Fig. 3bJ) , fibril formation of fibronectin (Fig. 3bK) , and their colocalization (Fig. 3bL) . 

Effect of Overexpression of the Akt1(K179M) Gene on Enhanced tTgase Expression and Extracellular Activity in Subconjunctival Fibroblasts

To investigate this possibility further, an Akt dominant negative mutant gene, Akt1(K179M), was transiently overexpressed in subconjunctival fibroblasts. As shown in Figure 4a , the expression of AKT1(K179M) in subconjunctival fibroblast by transient transfection was increased in a time-dependent manner. Figure 4bshows that decreased amounts of tTgase were present in the lysates of TGF-β2-stimulated cells transfected with Akt1(K179M) (lane 3), relative to TGF-β2-stimulated cells transfected with vector only (lane 2). These results confirm that the decreased expression of tTgase was coincident with inhibition of Akt phosphorylation in subconjunctival fibroblasts. 

Regulation of Cell Surface tTgase Activity in Subconjunctival Fibroblasts by the PI3K-Akt Pathway

We further investigated the role of Akt in the activation of tTgase by quantifying tTgase activity via in situ cell surface tTgase activity. ¹⁶ In the quantitative assay for tTgase cell surface activity, the incorporation of biotinylated cadaverine into fibronectin increased significantly when cells were pretreated with TGF-β2 before seeding (Fig. 5) . Treatment of the TGF-β2-stimulated cells with LY294002 or tTgase siRNA decreased tTgase cell surface activity significantly, relative to cells treated with TGF-β2 only. The effect of Ly294002 was comparable to that of tTgase siRNA on cell-surface activity, further supporting the hypothesis that the PI3K-Akt pathway may play an important role in TGF-β2-induced expression of tTgase. U0126 had little if any effect on the activation of tTgase by TGF-β2. 

Discussion

Among the various growth factors involved in tissue repair of the conjunctiva, TGF-β has been shown to be a key modulator in tissue fibrosis. ^{17 18} Likewise, tTgase has been shown to be an important player in this process by binding and cross-linking several extracellular proteins, including fibronectin in particular. ^{19 20 21} tTgase functions not only to stabilize these proteins, but to facilitate cell adhesion and cell motility by catalyzing the aggregation or polymerization of proteins by calcium-dependent transamidation activity. ^{22 23} tTgase has been detected in various ocular tissues, including the ciliary body, iris, conjunctiva, and trabecular meshwork, where it may stabilize structural proteins and extracellular matrix–cell interactions. ^{24 25 26} Moreover, increased expression of tTgase has been associated with several pathologic conditions, including neurofibrillary tangles in Alzheimer disease ²⁷ and in renal fibrosis. ²⁸ tTgase has also been reported to play a role in cataractous lenses ²⁹ and in the retina of Royal College of Surgeons (RCS) rats, which develop hereditary retinal degeneration and light-induced retinal damage. ³⁰  

In the present study, we investigated the intracellular TGF-β signal transduction pathway involved in the expression and activity of tTgase in primary subconjunctival fibroblasts. Several prior studies have shown that the expression and activation of the tTgase is tightly coupled to the effects of TGF-β. For example, TGF-β has been reported to induce expression of tTgase in various cells including rabbit tracheal epithelial cells, human epidermal keratinocytes, rat hepatoma cell lines, and cultured human retinal pigment epithelial (RPE) cells. ^{31 32 33 34} Similar observations have been made with fibroblasts after treatment with TGF-β. ³⁵ Recently, it has been reported that TGF-β induces the expression of tTgase and the cross-linking of fibronectin in human trabecular meshwork. ²⁶ The authors speculated that induced expression of tTgase may lead to the aberrant increase of ECM in the trabecular meshwork and an increase in outflow resistance in glaucoma. In our present study, TGF-β stimulated the expression of tTgase and the associated fibronectin cross-linking activity in subconjunctival fibroblasts. We postulate that the induced expression of tTgase by TGF-β may lead to aberrant augmentation of ECM in subconjunctival fibroblast and play a role in reduction of the success rate in glaucoma filtration surgery. 

Various factors have been identified that induce tTgase in different cell types. For example, retinoic acid stimulates the expression and activation of tTgase in both human myeloblastic leukemia HL60 cells and mouse peritoneal macrophages. ^{36 37} TNF-α causes an increase in the formation of nonreducible HMW fibronectin complexes in lung endothelial cells, potentially because of enhanced tTgase activity on the endothelial cell surfaces. ³⁸ In human lens epithelial cells, in situ tTgase activity increased approximately threefold in response to oxidative stress and activated tTgase to catalyze the formation of water-insoluble dimmers of polymers of αB-crystalline, βb2-crystalline, and vimentin, providing evidence that tTgase may play a role in cataractogenesis. ³⁹ However, there have been few if any studies of how these factors modulate the expression of tTgase in these cells. 

In this study, we further investigated the signal transduction pathways involved in TGF-β-induced expression of tTgase. By screening the MAPK superfamily and PI3-K-Akt pathway, we found that TGF-β2 stimulated the phosphorylation of Akt and Erk1/2. Specific inhibitors, LY294002 (PI3K inhibitor) and U0126 (ERK1/2 inhibitor) has been shown to inhibit TGF-β2-induced Akt and Erk1/2 phosphorylation, respectively. However, only LY294002 decreased the TGF-β-induced expression of tTgase. Moreover, transfection with a dominant negative mutant AKT, Akt1(K179M), decreased the TGF-β2-induced expression of tTgase. These results indicate that the activation of Akt, but not Erk1/2, modulates TGF-β2-induced expression and activity of tTgase. Active PI3K generates several phosphoinositols, leading to Akt activation by phosphorylation at Thr308 and Ser473 by phosphoinositide-dependent kinase-1. ⁴⁰ Activated Akt is considered a key downstream survival factor, as it stimulates cell proliferation and inhibits apoptosis. The activation of PI3K-Akt pathway has been shown to play a diverse role in the fibroblast. PI3K at least partially facilitates the cell viability by β1 integrin interaction with ECM in response to mechanical forces in skin fibroblasts. ⁴¹ The PI3K-Akt pathway also activates the collagen synthesis by actively cell spreading plates and platelet-derived growth factor. ⁴²  

Antonyak et al. ⁴³ recently showed that retinoic acid activates the signaling molecule PI3K, and that this activation is essential for the induction of expression and subsequent activation of tTgase by retinoic acid. They also showed that both RAR transcription and Akt activation may be involved in the cell survival process through tTgase. Our results showed that TGF-β2 stimulated the phosphorylation of Akt in a delayed manner, with maximum detectable induction at 24 hours. This pattern is distinct from retinoic acid induction of Akt phosphorylation. A recent report suggests that TGF-β induces a delayed activation response of PI3 kinase in epithelial cells and that a signaling intermediate between ligand-bound TGF-β receptors and PI3K may exist. ⁴⁴ Therefore, TGF-β-induced expression of tTgase by the PI3-K-Akt pathway is clearly different from retinoic acid–induced expression of tTgase, and further studies are warranted to investigate the signaling intermediate between ligand-bound TGF-β and PI3K-Akt. We further demonstrated that cell surface tTgase activity was modulated by the PI3-K-Akt pathway, by showing that the cross-linking of fibronectin was decreased by LY294002 and tTgase siRNA in a cell surface activity assay. We observed the phosphorylation of Smad 2/3, a transcriptional activator, in subconjunctival fibroblasts treated with TGF-β. Treating cells with PI3Kinase inhibitor, however, did not affect this activation (data not shown), indicating that the Smad pathway may not be directly involved in TGF-β-induced expression of tTgase. 

In conclusion, TGF-β2 activated the PI3K-Akt pathway, and this activation was essential for the expression and activity of tTgase in subconjunctival fibroblasts. Our results indicate a novel biological function of the PI3K-Akt pathway in subconjunctival fibroblasts. Elevated expression and activity of tTgase may play an important role in the pathogenesis of diseases related to wound healing and fibrogenic reactions in subconjunctival fibroblasts. These results also raise the possibility that specific inhibitors of the PI3K-Akt cascade may have beneficial effects in preventing pathogenic fibrosis in subconjunctival fibroblasts. 

²  Contributed equally to the work and therefore should be considered equivalent authors.

 Supported by Grant R01-2003-000-11649 from the Korean Science and Engineering Foundation.

 Submitted for publication September 28, 2006; revised November 30, 2006; accepted February 14, 2007.

 Disclosure: S.-A. Jung, None; H.K. Lee, None; J.S. Yoon, None; S.-J. Kim, None; C.Y. Kim, None; H. Song, None; K.-C. Hwang, None; J.B. Lee, None; J.H. Lee, 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.

 

Enhanced expression of tTgase by TGF-β2 in human subconjunctival fibroblasts. Subconjunctival fibroblast cells grown in complete growth medium were placed in serum-free medium overnight and were then treated with TGF-β2. (a) Time response effects: Cells incubated with 10 ng/mL of TGF-β2 in serum-free medium were harvested at the indicated time points and analyzed by Western blot. (b) Dose–response effects: After incubation with various concentrations of TGF-β2 for 48 hours, the cells were extracted and analyzed by Western blot analysis. Results are representative of those in three repeated experiments.

Figure 2.

 

Phosphorylation of Akt and ERK 1/2 induced by TGF-β2 in human subconjunctival fibroblasts. Subconjunctival fibroblast cells grown in complete growth medium were placed in serum-free medium overnight and were then treated with 10 ng/mL TGF-β2 at the indicated times. Activation and expression levels of ERK1/2, p38 MAPK, JNK, Akt, and PI3K were examined in each of the cell lysates. Western blot analysis with phosphospecific antibodies was used to measure the activities of the proteins. After initial analysis, the blots were stripped and reprobed with antibodies for total protein to assess expression levels of each of these proteins. Results are representative of those in three repeated experiments.

Figure 3.

 

(a) LY294002 inhibited TGF-β2-induced expression of tTgase in human subconjunctival fibroblasts. Subconjunctival fibroblasts grown in complete growth medium were placed in serum-free medium overnight and were then placed in a serum-free medium containing 6 μM LY294002 or 10 μM U0126±10 ng/mL TGF-β2 for 2 days, lysed, and analyzed by Western blot to determine expression levels of tTgase. Results are representative of those in three repeated experiments. (b) Double-immunofluorescence analysis of in situ tTgase activity, as shown by tTgase-mediated fluorescein cadaverine (green) incorporation and fibronectin (red) fibril formation. The colocalization of fluorescein cadaverine and fibronectin resulted in a mixed bright yellow with double-filter analysis. In nontreated control cells, fluorescein cadaverine (b A) and fibronectin (b B) were sparse, and only very small yellow spots were observed between the cells after superimposition of the images (b C). In TGF-β2-treated cells, staining for fluorescein cadaverine (b D) and fibronectin (b E) was markedly augmented, and prominent yellow spots were visible between the cells (b F), indicating an increase in the colocalization of fluorescein cadaverine and fibronectin. Staining of fluorescein cadaverine (b G) and fibronectin fibrils (b H), and colocalization (b I) were markedly decreased after exposure of TGF-β2-treated cells to LY294002. Exposure of the cells to U0126 did not alter staining of fluorescein cadaverine (b J) or fibronectin fibrils (b K) and colocalization (b L). Original magnification, ×400.

Figure 4.

 

(a) Expression of AKT1(K179M) in subconjunctival fibroblast by transient transfection was confirmed by Western blot analysis. An anti-His antibody was used to detect the expression of AKT1(K179M). (b) Inhibition of TGF-β2-induced expression of tTgase by transient transfection of Akt1(K179M) in human subconjunctival fibroblasts. Subconjunctival fibroblast cells were transiently transfected with an Akt dominant negative mutant, Akt1(K179M), in complete growth medium, placed in a serum-free medium containing 10 ng/mL TGF-β2 for 48 hours, and then lysed. The lysates from vector-transfected control cells (lane 1), vector-transfected cells treated with TGF-β2 (lane 2), and Akt1(K179M)-transfected cells treated with TGF-β2 (lane 3) were extracted and fractionated by SDS-PAGE. Results are representative of three independent experiments. Data represent the mean ± SD of results in three independent experiments.

Figure 5.

 

LY294002 or tTgase antisense siRNA induced inhibition of cell surface tTgase activity in TGF-β2-treated human subconjunctival fibroblasts. Subconjunctival fibroblast cells grown in complete growth medium were placed in serum-free medium overnight and were then placed in a serum-free medium containing either 6 μM LY294002, 10 μM U0126, or tTgase siRNA in the presence of 10 ng/mL TGF-β2 for 24 hours. These subconjunctival fibroblast cells were plated (2 × 10⁴ cells/well) in serum-free medium (DMEM) in the presence of 0.1 mM biotinylated cadaverine. Cells were allowed to incubate on the fibronectin-coated plates for 1 hour at 37°C. Reactions were stopped by washing cells with PBS containing 3 mM EDTA. Color development was determined with an ELISA plate reader at 450 nm. Data represent the mean ± SD of results in three independent experiments.