Keratinocyte Transglutaminase in Proliferative Vitreoretinopathy

Abstract

purpose. Proliferative vitreoretinopathy (PVR) is an excessive wound–healing process and the major complication in rhegmatogenous retinal detachment. The present study was designed to investigate the expression of keratinocyte transglutaminase (kTgase) in PVR membranes and retinal pigment epithelial (RPE) cells and to evaluate its expression in response to growth factors known to be increased in PVR disease.

methods. Distribution of kTgase and its relation to fibronectin have been investigated immunohistochemically. PVR membranes and native and cultured RPE cells were analyzed by RT-PCR for the presence of kTgase mRNA. In vitro, RPE cells were treated with transforming growth factor (TGF)-β2, basic fibroblast growth factor, interleukin-6, and interleukin-1β. Expression of kTgase was studied by Northern and Western blot analysis. The effect of connective tissue growth factor (CTGF) silencing on the TGF-β2–modulated expression of kTgase was investigated by transfection with CTGF small interfering (si)RNA before TGF-β2 treatment.

results. mRNA expression of kTgase was present in all PVR membranes. Immunohistochemical staining for kTgase showed an inhomogeneous pattern with localized accumulation and little colocalization with fibronectin. Although native RPE cells contained only a basal level of kTgase mRNA, the expression of kTgase was increased under culture conditions and was further enhanced by TGF-β2 treatment. Silencing of CTGF expression did not attenuate the TGF-β2–mediated induction of kTgase.

conclusions. The findings demonstrate that kTgase is present in PVR membranes. Its amount is related to the differentiation state of RPE cells and stimulation by TGF-β2. TGF-β2–mediated increase seems to be independent of CTGF.

Proliferative vitreoretinopathy (PVR) is characterized by the development of epiretinal and subretinal fibrocellular scarlike membranes containing modified retinal pigment epithelium (RPE) cells among others. ^{1 2} We have recently demonstrated that tissue transglutaminase (tTgase) is present and active in PVR membranes and may contribute to excessive accumulation of extracellular matrix (ECM) by tTgase-induced cross-linking of ECM proteins in PVR membranes. ³ The amount and activity of this enzyme appeared to be related to the differentiation state of the RPE cells and their stimulation by transforming growth factor (TGF)-β2, a growth factor known to be increased in the vitreous of PVR. ⁴  

The transglutaminase (Tgase) family of enzymes (EC 2.3.2.13) are calcium dependent ^{5 6 7} and participate in many biologic processes involving cross-linking proteins into large macromolecular assemblies. They are responsible for blood clotting, ⁷ apoptosis, ⁸ seminal vesicle coagulation, ⁹ cataract formation, ¹⁰ extracellular matrix and bone formation, ¹¹ cornified envelope formation, and barrier function in stratified squamous epithelia. ^{12 13} Nine isozymes are known in humans ^{5 14} : Tgase 1 (keratinocyte transglutaminase [kTgase], mostly membrane bound, expressed in epithelia), Tgase 2 (tissue transglutaminase [tTgase], soluble, ubiquitously expressed), Tgase 3 (epidermal transglutaminase, soluble, expressed mostly in epithelia), Tgase 4 (soluble, expressed mostly in prostate), Tgase 5 (ubiquitous, except for brain), Tgase 6 (ubiquitous, physiological significance unknown), Tgase7 (tissue distribution and physiological function unknown), band 4.2 (expressed in erythrocytes, membrane skeletal component), and factor XIIIa (soluble, circulating blood cells). 

The keratinocyte transglutaminase, or kTgase, is an enzyme expressed during terminal differentiation of epidermal keratinocytes. Many aspects of its biochemical properties and substrate proclivities are now well understood. ^{12 15 16 17} In epidermal differentiation, kTgase catalyzes irreversible ε-γ-(glutamyl) lysine cross-links of proteins to stabilize the cell envelope at the periphery of cornified cells. ^{18 19 20} Experiments with knockout mice revealed that kTgase is essential for the distribution of the cell envelope precursor protein at the cell periphery and that the function of kTgase cannot be compensated for by other Tgase isozymes. ²¹ kTgase is also involved in scar tissue formation in severe ocular surface diseases such as Stevens–Johnson syndrome, ²² ocular cicatricial pemphigoid, and chemical injury. ²³  

PVR is characterized by the formation of scarlike fibrocellular membranes on the retinal surface, in the vitreous, and in the subretinal space. ^{1 2} The precise pathogenic mechanism involved in the formation of epiretinal and subretinal membranes is not completely understood. 

Whereas in the healthy adult eye the RPE forms a nonproliferating monolayer of polarized, stationary cells essential for the maintenance and survival of the photoreceptors, ²⁴ after retinal detachment RPE cells become disseminated from their normal site on Bruch membrane and are dispersed to multiple loci on the neuroretina and in the vitreous. The dislodged cells dedifferentiate and exhibit a pseudometaplastic transformation into fibroblastlike cells that are actively dividing and migrating. ²⁵ Because of the breakdown of the blood–retinal barrier after retinal injury, cytokines including TGF-β2, basic fibroblast growth factor, interleukin (IL)-6, and IL-1β, among others, are increased in the vitreous cavity of patients with PVR. ⁴ They are thought to further stimulate the pseudometaplastic transformation of dislodged RPE cells into fibroblastlike cells and to increase RPE cell proliferation and migration. ²⁵ These processes are believed to be the key events in the onset of PVR. ^{1 2 25 26}  

When human RPE cells are cultured on plastic, they escape growth arrest and fail to maintain a differentiated morphology. They rapidly dedifferentiate at the molecular level, and proteins associated with highly specialized functions of the RPE, such as interaction with photoreceptor cells, become undetectable, ^{27 28 29} though they display a strong shift toward increased expression of proteins associated with cell adhesion, motility, cell shape, and proliferation. ^{27 30 31} This provides a well-accepted in vitro model for the fibroblastlike phenotype of RPE cells as found in PVR. ^{25 32 33 34 35 36 37} In a previous study, we noted an association of RPE dedifferentiation in vitro and upregulation of tTgase expression. ³ We also found tTgase activity and the presence of the enzyme in PVR membranes. ³  

Previous studies have shown that severe ocular surface diseases, which are characterized by chronic cicatricial phases, ^{22 23} are accompanied by a significant increase of kTgase expression. In view of this, we investigated the possible role of kTgase as a representative marker of irreversible cicatrization in PVR disease, which is also characterized by excessive scar tissue formation. We evaluated kTgase expression in PVR membranes and its expression during RPE cell dedifferentiation and after growth factor treatment in vitro. 

Materials and Methods

Tissue Samples

Twenty-five samples of epiretinal or subretinal PVR membranes were obtained from patients undergoing vitreoretinal surgery for PVR performed at the Department of Ophthalmology of the Ludwig-Maximilians-University in Munich. Methods for securing human tissue were humane, included proper consent and approval, and complied with the Declaration of Helsinki. All patients with PVR experienced previous rhegmatogenous retinal detachment. 

Operations were performed by different surgeons using the same technique. Conventional vitreous surgery was performed with a three-port system. Epiretinal and subretinal membranes were separated from the retina by peeling whole-mount tissues. 

Membranes were put into phosphate-buffered saline (PBS, pH 7.4) intraoperatively and either snap-frozen in liquid nitrogen for mRNA extraction or mounted in OCT mounting media (Merck, Darmstadt, Germany) and then stored in liquid nitrogen for cryostat sections. 

Immunohistochemical Staining of Tissue Sections

Immunohistochemical double staining for extracellular kTgase and fibronectin was performed in sections obtained from 12 PVR membranes. Unfixed PVR membranes were cut at a thickness of 8 μm. After washing in Tris-buffered saline (TBS, pH 7.2–7.4) and preincubation with dry-milk solution (Blotto; Merck) to minimize nonspecific staining, the sections were incubated for 2 hours at room temperature (RT) with mouse anti–keratinocyte transglutaminase (Paesel & Lorei, Duisburg, Germany) and rabbit anti–fibronectin (Sigma, Deisenhofen, Germany). All used antibodies were diluted 1:100 in TBS containing 3% bovine serum albumin (BSA). After washing in TBS, the sections were incubated with goat anti–mouse IgG Cy-3 and swine anti–rabbit IgG Cy-2 (Dianova, Hamburg, Germany) diluted 1:100 in blocking buffer for 2 hours at RT. 

Control sections were incubated with BSA-TBS replacing the primary antibody. A fluorescence microscope (Leica, Wetzlar, Germany) was used to study the stained sections. 

Isolation of Human RPE Cells

Sixteen human donor eyes were obtained from the Munich University Hospital Eye Bank and processed within 4 to 16 hours of death. Donors ranged in age from 15 to 73 years. None of the donors had known history of eye disease. Methods for securing human tissue were humane, included proper consent and approval, and complied with the Declaration of Helsinki. Human RPE cells were harvested after the procedure, as has been described previously. ^{3 35} In brief, whole eyes were thoroughly cleansed in 0.9% NaCl solution, immersed in 5% polyvinyl pyrrolidone iodine, and rinsed again in the sodium-chloride solution. The anterior segment of each donor eye was removed, and the posterior poles were examined with the aid of a binocular stereomicroscope to confirm the absence of gross retinal disease. Next, the neural retinas were carefully peeled from the RPE–choroid–sclera using fine forceps. The eyecup was rinsed with Ca²⁺- and Mg²⁺-free Hanks balanced salt solution and treated with 0.25% trypsin (Gibco, Karlsruhe, Germany) for 1 hour at 37°C. Trypsin was aspirated, and the eyecup was filled with Dulbecco modified Eagle medium (DMEM; Biochrom, Berlin, Germany) supplemented with 20% fetal calf serum (FCS; Biochrom). The medium was gently agitated with a pipette, and the RPE was released into the media without damage to Bruch membrane. 

For RT-PCR analysis, the RPE cells from six donors were released from Bruch membrane by gently pipetting of 0.5× PBS solution into the eye. Suspended RPE cells were transferred to a 1.5-mL microcentrifuge tube and were centrifuged for 5 minutes at 129g. After centrifugation, the supernatant was removed and replaced by RNA extraction solution. 

Human RPE Cell Culture

The RPE cell suspension was transferred to a 50-mL flask (Falcon, Wiesbaden, Germany) containing 20 mL DMEM (Biochrom) supplemented with 20% FCS (Biochrom) and maintained at 37°C and 5% carbon dioxide. Epithelial origin was confirmed by immunohistochemical staining for cytokeratin ³⁸ with the use of a pan-cytokeratin antibody (Sigma; data not shown). The cells were tested and found free of contaminating macrophages (anti-CD11; Sigma) and endothelial cells (anti–von Willebrand factor; Sigma; data not shown). 

For growth factor experiments, second- to fifth-passage RPE cells were grown to confluence. RPE cells were then washed, incubated overnight in serum-free medium, and subsequently incubated in serum-free DMEM supplemented with 2.0 ng/mL TGF-β2 (R&D Systems, Wiesbaden, Germany), 200 pg/mL basic fibroblast growth factor (bFGF; PeproTech, Rocky Hill, NJ), 320 pg/mL IL-6; PeproTech), and 5 pg/mL IL-1β (PeproTech) for 24 hours. Controls were incubated under identical conditions without growth factors in the medium. 

RT-PCR of PVR Membranes and Cultured Human RPE Cells

Total mRNA of 13 PVR membranes was extracted using a micro-RNA kit (pepGOLD RNAPure; Peqlab, Erlangen, Germany). Total RNA from native human RPE cells and RPE cells grown in 10-cm dishes, as described, was extracted by the guanidinium thiocyanate–phenol–chloroform extraction method (Stratagene, Heidelberg, Germany). After confirming the structural integrity of the total RNA by electrophoresis on 1% agarose gels and subsequent staining with 0.5 μmol/mL ethidium bromide, RNA samples were treated with 3 U RQ RNase-free DNAse (Promega, Madison, WI) for 35 minutes at 37°C to remove traces of contaminating genomic DNA. The content of RNA was measured by photometric measurement, and the RNA concentration was adjusted. With the use of Moloney murine leukemia virus (MMLV) reverse transcriptase and oligo(dT)-17 primer (Gibco), first-strand complementary DNA (cDNA) was prepared from total RNA. Quality of RNA and cDNA synthesis was proven by amplification of the housekeeping gene glyceraldehyde-3-phosphatedehydrogenase (GAPDH). 

PCR of the same quantity of total cDNA was performed in a total volume of 50 μL with 1 U native Taq polymerase (Eppendorf, Hamburg, Germany). The following primer pairs (Metabion, Munich, Germany) were used: (1) kTgase: forward, 5′-AAGAGACTAGCAGTGGCATCTTCTG-3′; reverse, 5′- CCTGAGACATTGAGCAGCATGG-3′; product size, 625 bp; annealing temperature, 58.9°C; (2) GAPDH: forward, 5′-CCTGCTTCACCACCTTCTTG-3′; reverse, 5′-CATCATCTCTGCCCCCTCTG-3′; product size, 437 bp; annealing temperature, 59.7°C; (3) RPE-65: forward, 5′-GTTTCTGATTGTGGATCTC-3′; reverse, 5′-GGGATGTTAATCTCCACTTC-3′; product size, 600 bp; annealing temperature 55.0°C. ³⁹ PCR was started with a hot start: 10 minutes for 94°C to denature DNA, followed by 36 cycles of 1-minute melting at 94°C, 1-minute annealing at the respective annealing temperature, and 2-minute extension at 72°C in a thermocycler (Mastercycler Gradient; Eppendorf). After the last cycle, the polymerization step was extended for another 10 minutes to complete all strands. Each PCR reaction was repeated at least twice. The specificity of the PCR product was analyzed by sequencing (Sequiserve, Vaterstetten, Germany). PCR performed on each sample of RNA that had not been reverse transcribed to cDNA was used as negative control and showed no amplified product. For semiquantitative RT-PCR, the number of cycles was optimized by checking amplification after each cycle from cycles 23 to 36 for kTgase and RPE-65 and from 20 to 33 for GAPDH. This showed that the 30th cycle was in the geometric phase for kTgase and GAPDH and for RPE-65 and GAPDH. Band intensity was measured with a workstation (LAS-1000 Imager; RayTest, Pforzheim, Germany). Quantification was performed with the appropriate software (AIDA; RayTest). The final amount of PCR product was expressed as the ratio of the kTgase gene amplified to that of the GAPDH gene. 

RNA Isolation and Northern Blot Analysis

Total RNA was isolated from confluent RPE cultures in 10-cm Petri dishes using the guanidinium thiocyanate-phenol-chloroform extraction method (RNA isolation kit; Stratagene, Heidelberg, Germany). Total RNA (3 μg/lane) was denatured and size-fractionated by gel electrophoresis in 1% agarose gels containing 2.2 M formaldehyde. RNA was then vacuum blotted onto a nylon membrane (Roche, Basel, Switzerland) and cross-linked (1600 μJ; Stratalinker; Stratagene). To assess the amount and quality of the RNA, the membrane was stained with methylene blue, and images were taken (LAS-1000 Imager; RayTest). 

Riboprobes for analysis of kTgase mRNA expression were synthesized by PCR using the same conditions and primer pairs as described for RT-PCR analysis. For synthesis of connective tissue growth factor (CTGF) riboprobes, the following primer pair was used: forward, 5′-AGGGCCTCTTCTGTGACTTC-3′; reverse, 5′-GGCCAAACGTGTCTTCCAGT-3′; product size, 336 bp; annealing temperature, 59.1°C. The T7-promoter sequence was added to the 5′ end of the downstream primer each time. PCR amplification products were separated by agarose gel electrophoresis and stained with ethidium bromide for visualization. Sequences were confirmed by automated DNA sequencing (Sequiserve). After purification (PCR Purification Kit; Qiagen, Hilden, Germany), 1 μg DNA was used as template for in vitro transcription with the digoxigenin labeling RNA kit (Roche). Labeling efficiency was confirmed by direct detection of the labeled RNA probe with anti–digoxigenin-alkaline phosphatase (Roche). 

Prehybridization, hybridization, and chemiluminescence detection of the digoxigenin riboprobe were performed as described previously. ^{3 40} In brief, after hybridization, the membrane was washed twice with 2× SSC, 0.1% sodium dodecyl sulfate (SDS) at RT followed by two washes in 0.1× SSC, 0.1% SDS for 15 minutes at 68°C. After hybridization and posthybridization washes, the membrane was washed for 5 minutes in washing buffer (100 mM maleic acid, 150 mM NaCl, pH 7.5, 0.3% Tween 20) and was incubated for 60 minutes in blocking solution. Blocking solution contained 100 mM maleic acid (pH 7.5), 150 mM NaCl, and 1% blocking reagent (Roche). Anti–digoxigenin alkaline phosphatase (Roche) was diluted 1:10,000 in blocking solution and was used to incubate the membrane for 30 minutes. The membrane was then washed four times (15 minutes each time) in washing buffer and was equilibrated in detection buffer (100 mM Tris-HCl; 100 mM NaCl; pH 9.5) for 10 minutes. For chemiluminescence detection, alkaline phosphate substrate (CDP-Star; Roche) was diluted 1:100 in detection buffer and was used to incubate the filter for 5 minutes at RT. After air drying, the semidry membrane was sealed in a plastic bag. Chemiluminescence was detected with the workstation (LAS-1000 Imager; RayTest), with exposure times ranging from 10 minutes to 1 hour. Chemiluminescent signal quantification was performed with the software package (AIDA; RayTest). 

Western Blot of kTgase

Cells grown in 60-mm tissue culture dishes were washed twice with PBS, collected, and lysed in NP-40 (150 mM NaCl, 50 mM Tris, pH 8.0, 1% NP-40) cell lysis buffer. Samples for gel analysis were boiled for 5 minutes, and protein content was measured with BCA protein assay reagent (Pierce, Rockford, IL). Proteins were loaded (12 μg/lane) and separated by electrophoresis with a 5% SDS-PAGE stacking gel and an 8% SDS-PAGE separating gel. ⁴¹ After gel electrophoresis, the proteins were transferred with semidry blotting onto a polyvinylidene diflouride (PVDF) membrane (Roche). The membrane was incubated for 1 hour with PBS containing 0.1% Tween 20 (PBST; pH 7.2) and 5% PBS. The primary antibody antikeratinocyte transglutaminase (1:2000; Paesel & Lorei) was then added and allowed to react overnight at RT. After the membrane was washed three times in PBST, it was incubated with an alkaline phosphatase–conjugated swine-anti–mouse antibody (Dianova; diluted 1:20,000) for 30 minutes Visualization of the alkaline phosphatase was achieved with chemiluminescence. Alkaline phosphate substrate (CDP-Star; Roche) was diluted 1:100 in detection buffer, and the filter was incubated for 5 minutes at RT. After air drying, the semidry membrane was sealed in a plastic bag. Chemiluminescence was detected with the workstation (LAS-1000 Imager; RayTest) at exposure times of 1 minute to 5 minutes, and quantification of chemiluminescence was performed (AIDA; RayTest). 

Generation and Transfection of siRNA

Small interfering (si)RNAs were designed according to the recommendations published by Elbashir et al. ^{42 43} Target sequences for the human CTGF siRNA were designed with Web-based criteria and generated with an siRNA construction kit (Silencer; Ambion, Austin, TX). Different CTGF siRNAs were tested in initial transfection and subsequent Northern blot experiments (data not shown). Best results were obtained by transfection of 10 nM CTGF siRNA with the transfection reagent, according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA). Primers used to generate this CTGF siRNA were CTGF 5′-AACAGTTGGCTCTAATCATAGCCTGTCTC-3′ (sense) and 5′-AACTATGATTAGAGCCAACTGCCTGTCTC-3′ (antisense). Maximum silencing was reached no more than 3 hours after transfection, and the effect lasted until at least 72 hours after transfection (data not shown). To assess the influence of CTGF on TGF-β2–mediated induction of kTgase, cells were seeded as previously described, transfected with 10 nM CTGF siRNA, and supplemented with medium containing TGF-β2 to a final concentration of 2.0 ng/mL after 4 hours. Cells were incubated in this manner for 48 hours before they were harvested for RNA isolation. At least three independent experiments were performed. 

Results

kTgase mRNA and Protein

PVR Membranes.

On RT-PCR, kTgase mRNA was observed in all investigated epiretinal and subretinal PVR membranes (Fig. 1) . The actual size of the PCR product of kTgase was close to the theoretically expected value (on the basis of the 625-bp primer position). The sequence was confirmed by automated DNA sequencing (data not shown). 

Immunohistochemical staining revealed specific staining for kTgase in all sections of PVR membranes studied (Figs. 2A 2D 2G) . Staining for kTgase showed areas of focal accumulation that appeared in punctuate or fusiform patterns (Figs. 2A 2D 2G) . Staining for fibronectin (FN) was present throughout the entire ECM of the PVR membrane (Figs. 2B 2E 2G) . In some regions, the rim of the membrane showed more intense staining for FN than the central area of the PVR membrane. As visualized by double staining, FN and kTgase showed little colocalization (Figs. 2C 2F 2I) . The extracellular matrix was invariably negative for kTgase (Figs. 2C 2F 2I) . 

Cell Culture of Human RPE Cells.

PVR membranes contain a predominance of proliferative and migratory RPE cells and other cell types at various stages of transdifferentiation. ⁴⁴ To determine whether kTgase expression in RPE cells is modulated under these conditions, kTgase expression was investigated in native (differentiated) RPE cells and in cultured (dedifferentiated) RPE cells, which are proliferative and migratory (i.e., wound repair phenotype). ⁴⁴ Semiquantitative RT-PCR analysis for RPE-65, a microsomal protein synthesized by differentiated RPE cells in vivo, ²⁸ was used to evaluate the biochemical differentiation status of investigated RPE cells (Fig. 3A) . In native RPE cells, the 600-bp amplicons representing RPE-65 mRNA were present, whereas no RPE-65 transcript was detectable in cultured RPE cells of passages 3 to 5. 

RT-PCR from native and cultured human RPE cells revealed the presence of kTgase mRNA (625 bp) in both cell types (Fig. 3B) . Semiquantitative analysis showed that the expression of kTgase mRNA in cultured RPE cells was five times higher than in freshly isolated native RPE cells (Fig. 3B) . All investigated RPE cell preparations and cell cultures showed similar results. PCR performed on negative control samples, when the reverse transcriptase step was omitted, produced negative results (data not shown). 

kTgase Induction by TGF-β2

Treatment of cultured RPE cells with TGF-β2 markedly increased the level of kTgase mRNA (Fig. 4A) . Levels of kTgase mRNA in TGF-β2–treated RPE cells were approximately 4.8 ± 1.3 (SD)–fold higher than those detected in the untreated control cells, whereas bFGF, IL-6, and IL-1β had no or little effect on kTgase expression in RPE cells. A representative blot is shown in Figure 4A . As shown by Western blot analysis, the induction of kTgase mRNA was paralleled by an increase of kTgase at the protein level. A single band was detected by Western blot analysis at the molecular mass of approximately 100 kDa (Fig. 4C) . Treatment with TGF-β2 increased the amount of kTgase in cultured RPE cells approximately 3.7 ± 1.1 (SD)–fold. Treatment of the cells with bFGF, IL-6, and IL-1β had no or marginal effect. A representative blot of the experiments, which were repeated at least three times, is shown in Figure 4C . 

CTGF Silencing and kTgase Expression

CTGF is a major regulator of ECM component expression and is likely a downstream mediator of TGF-β action. To evaluate whether kTgase expression in cultured human RPE cells is influenced by CTGF, CTGF expression was specifically suppressed by posttranscriptional gene silencing through RNA interference. For this purpose, duplexes of 21-nt RNA with a 2-nt 3′ overhang were introduced to cultured RPE cells by transfection. These short RNA duplexes specifically target complementary mRNA for degradation and are referred to as siRNAs. Twenty-four hours after transfection, gene silencing was documented by Northern blot analysis (Fig. 5) . As is typical of siRNA technology, a substantial downregulation—but not a complete knockout—of CTGF was achieved compared with controls. Posttrancriptional gene silencing with CTGF siRNA (siRNA-CTGF) reduced the 2.1-kb CTGF mRNA to approximately 20% of the expression level of the untreated controls. CTGF mRNA expression levels in control-transfected cells remained unchanged. 28S and 18S rRNA band intensities served as loading controls and were included for quantification. 

To test the possibility of an influence of CTGF on constitutive and TGF-β2–induced kTgase expression in cultured RPE cells, we conducted a series of silencing experiments with CTGF siRNA (Fig. 6) . Twenty-four hours after CTGF silencing, nontransfected controls (Co), control transfected (Co-L), and silenced CTGF expression (siRNA-CTGF; lanes 1–3) cells showed comparable kTgase expression levels. In addition, after 24 hours of incubation with 2.0 ng/mL TGF-β2, RPE cells transfected with CTGF siRNA before TGF-β2 (TGF-β2 + siRNA-CTGF) treatment showed an induction of kTgase mRNA expression levels similar to that of nontransfected TGF-β2–treated RPE cell cultures (TGF-β2; lanes 4–5). These results suggest that in cultured RPE cells, CTGF may not be involved in the regulation of kTgase expression at the constitutive expression level or after induction with TGF-β2. 

Discussion

Transglutaminases form a family of enzymes that have evolved for specialized functions such as protein cross-linking in hemostasis, apoptosis, cornified envelope formation, and wound healing. ^{5 6 15 19 45} Cross-linking activity is involved in disparate biologic phenomena, depending on the location of the target proteins. In the epidermis, the primarily membrane-bound kTgase can give rise to cross-linked envelopes in apoptotic and cornified epithelial cells. ^{15 19} Although the presence of kTgase has been well described in different stratified squamous epithelia, to the best of our knowledge this is the first report to demonstrate the presence of kTgase mRNA and protein in human PVR membranes and RPE cells. 

Cell biology experiments revealed that kTgase mRNA was hardly detectable by RT-PCR in native differentiated RPE cells. In contrast, cultured dedifferentiated RPE cells exhibited pronounced kTgase mRNA expression. Dedifferentiation of RPE cells to fibroblastlike cells is thought to be a key pathologic event in PVR disease. The breakdown of the blood–retinal barrier after retinal detachment further enhances this process and leads to elevated levels of several cytokines in the vitreous cavity. ^{4 46 47} We demonstrated that treatment of cultured RPE cells with TGF-β2 led to a further increase in kTgase expression, whereas the other cytokines tested had no or little effect. These results suggest that two conditions implicated in the pathogenesis of PVR—RPE dedifferentiation and elevation in TGF-β2 levels—may alter kTgase expression in the RPE. In a previous study, we found that treatment of cultured RPE cells with TGF-β2 also increased expression levels of tTgase, ³ another isozyme of the Tgase family, which is implicated in stabilizing the ECM formed during wound healing. The finding that TGF-β2 induces kTgase and tTgase in RPE cells further suggests that these two enzymes may play fundamental roles in the TGF-β2–mediated wound healing response in PVR. 

TGF-β is a pluripotent cytokine that regulates several biologic activities involved in the pathogenesis of PVR, including cell proliferation, ECM deposition, and cell migration. ⁴⁸ TGF-β promotes the deposition of ECM by inducing the expression of extracellular matrix components ⁴⁹ and decreasing the expression of matrix-degrading enzymes such as matrix metalloproteinases. ⁵⁰ It has been shown that CTGF, a matricellular protein that belongs to a family of immediate-early gene products, ⁵¹ seems to mediate at least some of the fibrotic effects of TGF-β by binding to TGF-β, thereby potentiating its binding to the TGF-β type II receptor or by prolonging ECM mRNA expression (for a review, see Leask and Abraham ⁵² ). In situ hybridization experiments demonstrated the presence of CTGF mRNA in fibroblastlike RPE cells in proliferative subretinal and epiretinal membranes. ⁵³ A direct link between TGF-β and CTGF expression has been found in astrocytes. In these cells, CTGF silencing repressed the TGF-β2–mediated upregulation of fibronectin, α1 type 1 collagen, α4 type 2 collagen, and tissue transglutaminase. ⁵⁴ However, as evidenced in the present study, CTGF in RPE cells appeared not to be involved in the regulation of kTgase expression. Cells with silenced CTGF expression and those with baseline CTGF levels showed comparable amounts of kTgase expression at the constitutive mRNA expression level. In addition, kTgase expression was similarly induced by TGF-β2 in cells with downregulated CTGF expression and in nontransfected controls, suggesting that in RPE cells the regulation of kTgase expression may be a direct target of TGF-β2 but may not be mediated by CTGF. 

Most of our current understanding of kTgase function and expression has arisen from studies in stratified squamous epithelia, where it appears in substantial amounts, typically in cells midway between the basal and the callus layers. In these cells, most kTgase is anchored to the plasma membrane, where it gives rise to the formation of irreversible cross-links between envelope proteins such as involucrin, cornifin, and loricrin beneath the plasma membrane. ^{15 55} We report for the first time the expression of kTgase in RPE cells and PVR membranes. As opposed to immunohistochemical findings made for tTgase in PVR, ³ staining for kTgase showed a different pattern. Although extracellular tTgase was evenly distributed throughout the entire PVR membrane with accentuation along the rim of the PVR membrane, ³ kTgase expression was characterized by a more inhomogeneous, punctuate, and fusiform staining pattern, reflecting a cell-associated location of the enzyme and showing little colocalization with fibronectin (FN). Even if the characteristics of RPE cells are different from those of keratinocytes, the punctuate and fusiform presence of kTgase in PVR membranes may suggest that, in a manner comparable to the role played in the epidermis, kTgase may cross-link cells to the surrounding ECM, thus supporting RPE cell adhesion to the newly deposited ECM. Recent studies demonstrated a comparable role in RPE adhesion for cell surface-associated tTgase. It has been found to promote adhesion and migration and to enhance the spreading of cells adhering to FN ^{3 56 57 58 59 60 61} as an integrin-associated adhesion coreceptor for FN. ⁵⁶ Clearly, the exact definition of ligand proteins for kTgase in PVR awaits further study. 

Although data regarding the expression of kTgase in tissues other than squamous epithelia are scarce, further support for a role of kTgase in extra-epidermal wound healing comes from a study on ulcerative colitis (UC). ⁶² UC is characterized by refractory inflammatory ulceration resulting from impaired wound healing. D’Argenio et al. ²⁸ found that kTgase expression is markedly reduced in patients with active UC. In contrast, PVR is a disease that entails excessive, abnormal wound healing. In keeping with this, increased expression of kTgase and tTgase ³ is found in PVR membranes. Although the role of kTgase in dedifferentiated RPE cells should be better defined in relation to its potential substrates, our finding that dedifferentiated and TGF-β2–treated RPE cells showed increased kTgase expression as a marker of increased cell adhesion and ECM stabilization remains of considerable interest. 

Together with the findings that tTgase is expressed at elevated levels in PVR membranes, ^{3 61} the results of the present study further support the concept that transglutaminases may play a key role in the abnormal wound healing in PVR because of the contribution to cell adhesion and excessive ECM accumulation. Therefore, it is tempting to speculate that the use of competitive inhibitors of kTgase and tTgase may offer new specific therapeutic strategies to prevent the formation of scarlike PVR membranes and to protect against this major complication in rhegmatogenous retinal detachment. 

 Supported by Deutsche Forschungsgemeinschaft Grant WE 2577/2–1.

 Submitted for publication March 14, 2006; revised June 7, 2006; accepted September 14, 2006.

 Disclosure: S.G. Priglinger, None; C.S. Alge, None; T.C. Kreutzer, None; A.S. Neubauer, None; C. Haritoglou, None; A. Kampik, None; U. Welge-Luessen, 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.

 

Agarose gel electrophoresis and ethidium bromide staining of PCR products amplified from epiretinal (lanes A–C) and subretinal (lanes D–F) PVR membranes with a primer specific for kTgase. Total mRNA (1 μg) was used as template for first-strand synthesis, and cDNA was then amplified by PCR by using the conditions described in Materials and Methods. Typical results are shown for experiments repeated at least twice. All investigated membranes showed similar results. PCR performed on negative controls, when the reverse transcriptase step was omitted, was negative (data not shown). MW: DNA standard lane is shown at the left of the gel.

Figure 2.

 

Micrographs showing immunohistochemical staining for kTgase (A, D, G) and fibronectin (FN) (B, E, H) in PVR membranes. Because of the staining of cellular elements (arrows), kTgase staining shows areas of focal accumulation and appears in punctuate or fusiform patterns (A, D, G), whereas FN staining is homogeneously distributed throughout the entire ECM of the PVR membrane with increased staining at the membrane’s margins (B, E, H). Immunohistochemical double staining of FN and kTgase reveals only little colocalization (C, F, I). Original magnifications, ×200.

Figure 3.

 

Agarose gel electrophoresis and ethidium bromide staining of PCR products amplified from native ¹ and dedifferentiated ² fibroblastlike human RPE cells of passage 3. mRNA expression of (A) RPE 65 and (B) kTgase in hRPE was measured by semiquantitative RT-PCR. The optical density of the kTgase PCR products was expressed as the ratio to the GAPDH amplicon of the same cDNA sample. Typical results are shown for experiments repeated three times. The number below each band shows the ratio of the optical density of the kTgase PCR product normalized to the GAPDH amplicon of the same cDNA. PCR performed on negative controls, when the reverse transcriptase step was omitted, were negative (data not shown). MW: DNA standard lane is shown at the left of the gel.

Figure 4.

 

(A) Northern blot analysis of kTgase mRNA confluent human RPE cells 24 hours after treatment with 2.0 ng/mL TGF-β2, 320 pg/mL IL-6, 5 pg/mL IL-1β, or 200 pg/mL bFGF. (B) Methylene blue staining of the 28S and 18S rRNA bands to demonstrate relative integrity and loading of RNA. (C) Western blot analysis of kTgase in RPE monolayers treated as described for Northern blot. Lysates from approximately equal amounts of protein (12 μg) were separated by SDS-PAGE and blotted for immunochemical detection of kTgase content. The number below each band shows the chemiluminescence measurement. Co, control; RDI, relative densitometric intensity (normalized to 28S rRNA).

Figure 5.

 

Silencing of CTGF expression in cultured human RPE cells by RNA interference. (A) Northern blot analysis of CTGF expression in nontransfected human RPE cells (Co), the same cell line transfected with vehicle and buffer alone instead of siRNA (Co-L), and cells transfected with a double-stranded siRNA complementary to CTGF (siRNA-CTGF). (B) Methylene blue staining of the 28S and 18S rRNA bands to demonstrate relative integrity and loading of RNA. RDI, relative densitometric intensity (normalized to 28S rRNA).

Figure 6.

 

Constitutive kTgase expression and TGF-β2–induced upregulation of kTgase in cultured human RPE cells are not blocked by CTGF siRNA. (A) Northern blot analysis of kTgase expression in nontransfected human RPE cells (Co), the same cell line transfected with vehicle and buffer alone instead of siRNA (Co-L), cells transfected with a double-stranded siRNA complementary to CTGF (siRNA-CTGF), nontransfected cells treated with 2.0 ng/mL TGF-β2 for 24 hours (TGF-β2), and the same cell line transfected with CTGF siRNA before TGF-β2 treatment (TGF-β2 + siRNA-CTGF). (B) Methylene blue staining of the 28S and 18S rRNA bands to demonstrate relative integrity and loading of RNA. RDI, relative densitometric intensity (normalized to 28S rRNA).