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Induction of Tissue Transglutaminase in the Trabecular Meshwork by TGF-ß1 and TGF-ß2

From the Department of Anatomy II, University of Erlangen-Nürnberg, Erlangen, Germany.

	Abstract

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PURPOSE. To study whether human trabecular meshwork (HTM) cells are capable of expressing and secreting tissue transglutaminase (tTgase), an enzyme cross-linking extracellular matrix (ECM) proteins, and whether tTgase and synthesis of cross-linked fibronectin are increased after treatment of HTM cells with transforming growth factor (TGF)-ß1 or -ß2.

METHODS. Anterior segments of six normal human eyes were stained with antibodies to tTgase. Tissues from three eyes were analyzed for tTgase using Western blot analysis. Monolayer cultures of HTM cells from eyes of five human donors were treated with 1.0 ng/ml TGF-ß1, -ß2, or 5 x 10^-7 M dexamethasone (DEX) for 12 to 96 hours. Induction of tTgase was investigated by Western and Northern blot analysis. External tTgase activity was measured by the ability to form polymerized fibronectin and the incorporation of biotinylated cadaverine into fibronectin.

RESULTS. Labeling for tTgase was observed throughout the entire HTM. Cultured HTM cells expressed tTgase intra- and extracellularly. Treatment of cultured HTM cells with TGF-ß1 and -ß2 increased the tTgase mRNA and protein levels, whereas DEX had no effect. TGF-ß–treated HTM cells showed a significant increase in polymerized and unpolymerized fibronectin. Incorporation of biotinylated cadaverine was markedly increased when HTM cells were treated with TGF-ß for 24 hours before seeding.

CONCLUSIONS. The enzyme tTgase is expressed in the HTM and is inducible by TGF-ß1 or -ß2 in cultured HTM cells. Extracellular tTgase is able to polymerize fibronectin. Increased levels of TGF-ß2 in the aqueous humor may lead to an increase of tTgase expression and activity in the HTM, causing an increase of irreversibly cross-linked ECM proteins. This mechanism might play a role for the increased outflow resistance seen in glaucomatous eyes.

	Introduction

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The human trabecular meshwork (HTM) is a specialized tissue that contributes to regulation of aqueous humor outflow and control of intraocular pressure (IOP). In most eyes with primary open angle glaucoma (POAG), IOP is increased because of a significant increase in resistance to aqueous outflow. This diminished outflow facility has been attributed to accumulation of extracellular matrix (ECM) material in the cribriform or juxtacanalicular region,^{1 2} which was first described as "plaque material" by Rohen and Witmer.¹ Subsequent studies showed that the "plaques" derive primarily from thickened sheaths of elastic fibers^{3 4} and consist of banded fibrillar elements embedded in different kinds of glycoproteins.⁵ The detailed nature of the plaques remains unknown. Most hypotheses about the pathogenesis of formation of glaucomatous changes in the HTM have focused either on the increased synthesis of the ECM^{1 2 5 6 7 8 9 10 11 12} or the decreased synthesis of metalloproteinases.^{7 8 9 13 14 15} Little attention has been paid to qualitative changes in HTM ECM. If the ECM were modified so as to make it more resistant to protease degradation, this would move the deposition–degradation balance toward accumulation. Such "stabilization" of the ECM in the HTM could have important consequences for aqueous humor outflow.

Transglutaminases are calcium-dependent enzymes that catalyze the posttranslational modification of proteins through an acyl transfer reaction between the -carboxamide group of a peptide-bound glutaminyl residue and various amines.¹⁶ Covalent cross-linking using -(-glutamyl) lysine bonds is stable and resistant to enzymatic, chemical, and mechanical disruption.¹⁶ Endopeptidases capable of hydrolyzing the -(-glutamyl) lysine cross-links formed by transglutaminases have not been described in vertebrates, and even lysosomes do not contain enzymes capable of splitting the -(-glutamyl) lysine bonds.^{17 18 19} Tissue transglutaminase (tTgase, type II) belongs to a wider family of transglutaminase enzymes, each of which has a distinct structure, location, and physiological function. Examples of this family include plasma Factor XIIIa involved in cross-linking fibrin during wound healing²⁰ and the keratinocyte enzyme involved in the terminal differentiation of keratinocytes.^{21 22}

tTgase is the most widespread member of this family and is present in many different cell types and tissues, with diverse functions.^{23 24 25} The enzyme plays a role in programmed cell death,¹⁹ cell adhesion,²⁶ and interaction between the cell and its ECM via the cross-linking of proteins, such as fibronectin,²² vitronectin,²⁷ laminin–nidogen complexes,^{23 28} and collagen type III.²⁹ All these components are present in the ECM of the trabecular meshwork (TM).^{30 31 32}

Because of their constant contact with the aqueous humor, the HTM cells are influenced by the substances contained therein. It has been shown that aqueous humor in a number of eyes with POAG contains increased amounts of TGF-ß2^{33 34} and that treatment with steroids can cause glaucoma.³⁵ We have therefore studied the influence of TGF-ß and dexamethasone (DEX) on tTgase synthesis by HTM cells in culture. The activity of extracellular tTgase was shown by the ability to cross-link fibronectin, an ECM component that is formed by HTM cells³⁰ and has been shown to be increased in glaucomatous eyes.³⁶

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Immunohistochemical staining for tTgase was performed in sections obtained from six human donor eyes (38, 42, 57, 63, 75, 83 years old). The eyes were obtained 6 to 16 hours postmortem and were fixed in 4% paraformaldehyde for 3 hours. For tissue culture studies TM was prepared from five human donor eyes (12, 49, 57, 57, 73 years old, obtained 4–8 hours postmortem) as described previously.³⁷ None of the donors had a known history of eye disease. Methods for securing human tissue were humane, included proper consent and approval, and complied with the Declaration of Helsinki.

Immunohistochemistry of Tissue Sections
Sagittal sections and serial tangential frozen sections, taken in a plane parallel to the inner wall of Schlemm’s canal (SC), were cut at a thickness of 10 to 14 µm, washed in Tris-buffered saline (TBS, pH 7.2–7.4), and preincubated with Blotto’s dry-milk solution (Merck, Darmstadt, Germany) to minimize nonspecific staining. Sections were incubated overnight at 4°C with mouse anti-tissue transglutaminase (Cub7402; Quartett, Berlin, Germany) diluted 1:100 in TBS containing 5% bovine serum albumin (BSA). After washing in TBS, the sections were incubated for 1 hour with biotinylated goat anti-mouse Igs (Dakopatts, Hamburg, Germany), diluted 1:200 in BSA-TBS and visualized with Cy3-conjugated streptavidin (1:50 for 1 hour; Dakopatts). Control sections were either incubated with BSA-TBS replacing the primary antibody or with a combination of 1:200 diluted primary antibody plus a fivefold weight excess of guinea-pig tTgase (Sigma-Aldrich, Deisenhofen, Germany).

Tissue Culture
Trabecular meshwork cells were grown and classified as described previously.^{37 38} Confluent HTM cells of passage 3 were incubated for 12, 24, 48, or 96 hours in serum-free Ham’s F-10 medium (Gibco-Life Science Technology, Karlsruhe, Germany) supplemented with either 1.0 ng/ml TGF-ß1 (Boehringer-Mannheim, Mannheim, Germany), 1.0 ng/ml TGF-ß2 (Boehringer-Mannheim), or 5 x 10^-7 M DEX (Sigma-Aldrich). The medium was changed every 24 hours, and TGF-ß1, -ß2, or DEX was added to the fresh medium. The treated cells were compared with cultures incubated under identical conditions, but without TGF-ß or DEX in the medium.

Immunohistochemistry of Cell Cultures
Intracellular tTgase.
HTM cells grown in four-well plastic-chamber slides were washed with phosphate-buffered saline (PBS, pH 7.4), fixed, and permeabilized by addition of 200 µl of 70% ethanol at -20°C for 15 minutes. tTgase was then detected by adding mouse anti-tTgase antibody (Cub7402; Quartett), diluted 1:200 in 0.1 M Tris-HCl, pH 7.4, followed by fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG (Dianova, Hamburg, Germany).

Extracellular tTgase and Fibronectin.
For detection of extracellular tTgase, confluent HTM cells grown in four-well plastic-chamber slides were incubated for 2.5 hours with serum-free Ham’s F10 medium containing 0.75 µg/ml monoclonal antibody to tTgase (Cub7402; Quartett). Cells were then washed in PBS and fixed in 4% paraformaldehyde in PBS. After blocking with BSA, cells were incubated with anti-mouse IgG-FITC for 2 hours at room temperature and then washed in PBS before mounting. For double staining of tTgase and fibronectin, cells were first stained for extracellular tTgase as above, but after blocking in 5% BSA, the cells were incubated for 15 hours at 4°C with rabbit anti-fibronectin antibody (Sigma-Aldrich) diluted 1:50 in blocking buffer. Samples were then washed with PBS and incubated with goat anti-mouse IgG-FITC and swine anti-rabbit IgG-tetramethylrhodamine isothiocyanate (TRITC) diluted 1:30 in blocking buffer for 2 hours at room temperature. Double staining was studied using a confocal laser microscope (Bio-Rad, London, UK).

RNA Isolation and Northern Blot Analysis
Total RNA was isolated from confluent HTM cultures in 35-mm Petri dishes using the guanidinium thiocinate-phenol-chloroform extraction method (RNA isolation kit; Stratagene, Heidelberg, Germany). Total RNA (15 µg/lane) was denaturated and size-fractionated by gel electrophoresis in 1% agarose gels containing 2.2 M formaldehyde. The RNA was then vacuum blotted onto a nylon membrane (Boehringer Mannheim) 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 with the Lumi-Imager (Boehringer Mannheim). Prehybridizations were performed at 68°C for 1 hour in Dig Easy Hyb (Boehringer Mannheim). Hybridizations were done at 68°C overnight in Dig Easy Hyb solution containing 50 ng/ml antisense riboprobe.

Riboprobes were synthezised from reverse transcription-polymerase chain reaction (RT-PCR) products obtained from HTM RNA using a T7 promoter tailed oligonucleotide. The cDNA was prepared from 0.5 µg total RNA from HTM cells by using 200 U SuperScript reverse transcriptase (Gibco Life Science Technology) and oligo(dT)-17 primer (Promega, Heidelberg, Germany). The RT reactions were diluted to 0.5 ml. The PCR was performed in a total volume of 50 µl using 1 U of native Taq DNA polymerase (Appligen-Oncor, Heidelberg, Germany), with the temperature profiles as follows: 36 cycles of 1 minute melting at 94°C, 1 minute annealing, and 2 minutes extension at 72°C. After the last cycle, the polymerization step was extended for a further 10 minutes so that all strands were completed. The primers were designed according to the published structures of the human genes for tTgase and fibronectin. In addition the reverse primer contained the sequence for the T7 promotor (underlined below). The sequences, position, product size and the annealing temperature of the primers were as follows: forward, 5'-ATTGGTCCAGACACCATGCG-3'and reverse, 5'-AATTGTAATACGACTCACTATAGGGCAACTTCCAGGTCCCTCGGAACATC-3' (positions, 3752–4288; product size, 537 bp; annealing temperature, 56.8°C) for fibronectin,³⁹ forward, 5'-CAGAACAGCAACCTTCTCATCGAG-3' and reverse, 5'-AATTGTAATACGACTCACTATAGGGCTTGGACTCCGTAAGGCAGTCAC-3' (positions, 1054–1881; product size, 784 bp; annealing temperature, 59.7°C) for tTgase.⁴⁰ All primers were purchased from MWG-Biotech (Ebersberg, Germany). After purification with a Qiagen (Hilden, Germany) PCR Purification Kit, PCR products were directly sequenced with fluorescent dideoxynucleotides on an automated sequencer (Applied Biosystems model 377; Perkin-Elmer, Überlingen, Germany). Using the digoxigenin-labeling RNA Kit from Boehringer Mannheim, 1 µg of DNA was used as a template for in vitro transcription. Digoxigenin labeling efficiency was checked by direct detection of the labeled RNA probe with anti-digoxigenin–alkaline phosphatase. After hybridization, the membrane was washed twice with 2x SSC, 0.1% sodium dodecyl sulfate (SDS) at room temperature, followed by two washes in 0.1x SSC, 0.1% SDS for 15 minutes at 68°C. After hybridization and posthybridizations 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 incubated for 60 minutes in blocking solution (100 mM maleic acid, 150 mM NaCl, pH 7.5, 1% blocking reagent; Boehringer Mannheim). Anti-digoxigenin-AP (Boehringer Mannheim) was diluted 1:10,000 in blocking solution and used to incubate the membrane for 30 minutes. The membrane was then washed four times, 15 minutes per wash, in washing buffer. The membrane was equilibrated in detection buffer (100 mM Tris-HCl, 100 mM NaCl, pH 9.5) for 10 minutes. For chemiluminescent detection, CDP-star (Boehringer Mannheim) was diluted 1:100 in detection buffer and used to incubate the filter for 5 minutes at room temperature. After air-drying, the semi-dry membrane was sealed in a plastic bag. Chemiluminescence was detected with the Lumi-Imager workstation (Boehringer Mannheim) with exposure times ranging from 10 minutes to 1 hour. Chemiluminescent signal quantification was performed with the Lumi Analyst software package (Boehringer Mannheim).

Western Blot of tTgase
Cells grown on tissue culture dishes were washed twice with PBS, pH 7.2, collected, and lysed in NP-40 (150 mM NaCl, 50 mM Tris, pH 8.0, 1% NP-40) sample buffer for gel analysis. The samples for gel analysis were boiled for 5 minutes, and protein content was measured using BCA protein assay reagent (Pierce, Rockford, IL). Proteins were loaded (2 µg/lane) and separated by electrophoresis using a 5% SDS-polyacrylamide stacking gel and a 8% SDS-polyacrylamide separating gel.⁴¹ After polyacrylamide gel electrophoresis (PAGE), the proteins were transferred with semi-dry blotting onto a PVDF membrane (Boehringer Mannheim). The membrane was incubated with PBS containing 0.1% Tween 20 (PBST, pH 7.2) and 5% BSA for 1 hour. The primary antibody (tTgase 1:2000, Cub7402; Quartett) was then added and allowed to react overnight at room temperature. After washing the membrane three times in PBST, an alkaline phosphatase–conjugated swine anti-mouse antibody (diluted 1:20,000; Dianova) was incubated with the membrane for 30 minutes. Visualization of the alkaline phosphatase was achieved using chemiluminescence. CDP-star was diluted 1:100 in detection buffer, and the filter was incubated for 5 minutes at room temperature. After air-drying, the semi-dry membrane was sealed in a plastic bag. Chemiluminescence was detected with the Lumi-Imager workstation with exposure times from 1 to 5 minutes. Quantification of chemiluminescence was performed with Lumi Analyst software (Boehringer Mannheim).

In additional experiments, tissue specimens (TM, sclera, cornea, ciliary process, iris) from three donor eyes (68, 46, 73 years old) were homogenized in ice-cold NP-40 sample buffer and used to perform Western blot analysis as described above.

Western Blot of Polymerized and Unpolymerized Fibronectin
For SDS-PAGE and Western blotting of fibronectin, cells were plated onto tissue culture plates (6-well plates), kept confluent for at least 7 days, and treated for 24 hours either with 1.0 ng/ml TGF-ß1 or -ß2. Cells were washed twice in PBS and then solubilized by addition of 200 µl of 2x strength Laemmli gel loading buffer (125 mM Tris-HCl, 20% glycerol, 4% SDS, 2% mercaptoethanol, and 10 mg/ml bromphenol blue). Solubilized cells were then boiled for 10 minutes, centrifuged, and subjected to SDS-PAGE using an 8% polyacrylamide resolving gel and 2.5% stacking gel by the method of Laemmli.⁴¹ After gel electrophoresis, the proteins were transferred with semi-dry blotting onto a PVDF membrane. To aid transfer of the polymerized protein, 75 µg/ml pronase (Sigma-Aldrich) was incorporated into the transfer buffer (10 mM Tris, 200 mM glycine, pH 8.0, without methanol), and the blotting paper was presoaked in this buffer before transfer. The membrane was incubated with PBST (pH 7.2) and 5% BSA for 1 hour. The primary antibody (rabbit anti-fibronectin, 1:2000; Sigma-Aldrich) was then added and allowed to react overnight at room temperature. The antibody binding was visualized as described above.

Incorporation of Biotinylated Cadaverine (BTC) into Fibronectin
Ttgase activity was measured by the incorporation of BTC into fibronectin.⁴² For this assay 96-well plates were precoated with plasma fibronectin (5 µg/ml; Sigma-Aldrich) incubated overnight at 4°C. Twenty-four hours before seeding, some HTM cells were treated with either 1.0 ng/ml TGF-ß1 or 1.0 ng/ml TGF-ß2. Untreated and TGF-ß–treated HTM cells were then plated at a density of 2 x 10⁵ cells/ml in 100 µl complete Dulbecco’s modified Eagle’s medium (DMEM) medium without serum in the presence of 0.1 mM BTC (Mobi-Tec, Göttingen, Germany). Cells were allowed to incubate in the fibronectin-coated plates for different time periods (0, 5, 10, 20, 40, 60, 90, or 120 minutes) at 37°C, after which time they were washed twice with PBS, pH 7.4, containing 3 mM EDTA. As a negative control, fibronectin-coated, 96-well plates were incubated with 100 µl DMEM medium without serum containing 0.1 mM BTC.

A detergent solution (100 µl) consisting of 0.1% deoxycholate in PBS, pH 7.4, containing 3 mM EDTA was then added to each well, and the mixture incubated with gentle shaking for 20 minutes. The supernatant was discarded, and the remaining fibronectin layer was washed three times with Tris-HCl, pH 7.4. Wells were then blocked with 3% BSA in Tris-HCl buffer for 30 minutes at 37°C and washed three times with Tris-HCl buffer, and then the incorporated BTC was revealed with a 1: 5000 dilution of Extravidin peroxidase conjugate (Sigma-Aldrich), which was incubated for 1 hour at 37°C. After washing three times with Tris-HCl, the fibronectin layer was incubated for 20 minutes at room temperature in 200 µl of substrate solution (a mixture of H₂O₂ and tetramethylbenzidine). Color development was stopped by adding 50 µl stop solution to each well. The optical density was determined by using a Molecular Devices (MWG-Biotech) ELISA reader set to 450 nm.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Immunohistochemical Staining for tTgase
In sagittal sections through the anterior segment, staining for tTgase was seen in the iris with intense labeling of the cells at the anterior surface of the iris stroma and the cells surrounding the vascular sheath (adventitial cells). Staining was also present in vascular endothelial cells as well as in stromal cells adjacent to the iris muscles (Fig. 1A ). Intense staining was also seen in the cells of trabeculum ciliare anterior to the ciliary muscle, connecting the uveal portion of the TM with the iris root. Within the ciliary muscle, the muscle cells were unstained (Fig. 1A) , whereas staining was present in the cells of the intermuscular connective tissue.

View larger version (112K): [in this window] [in a new window]   Figure 1. (A) Immunohistochemical staining of a sagittal section through the anterior segment of a human donor eye (age 63 years; magnification, x80). In the iris intense staining for tissue transglutaminase is seen in the cells forming the anterior surface of the iris stroma (arrowheads) and in the cells surrounding the vascular sheaths (arrow). The vascular endothelial cells are also stained. In the the ciliary muscle (CM) staining is confined to the cells of the trabeculum ciliare (small arrows) anterior to the ciliary muscle and cells in the intermuscular connective tissue, whereas the ciliary muscle cells are unstained. In the TM intense staining is seen in all layers. (B) Higher magnification of the staining in the TM as well in the inner and outer wall of Schlemm’s canal (SC) (magnification, x160). (C) Tangential section through the uveal (top) and corneoscleral (bottom) portion of the TM (age 57 years; magnification, x200). Note that all trabecular cells are intensely stained. Within the cell the entire cytoplasm is immunoreactive, whereas the nucleus is spared. (D) Control sections incubated with a combination of primary antibody and a fivefold excess of tTgase were unstained (magnification, x160).

In the HTM, intense staining for tTgase was present in essentially all portions as well as in the inner and outer walls of the SC (Fig. 1B) . Tangential sections, parallel to the inner wall of SC, revealed that staining was present in the cytoplasm of the trabecular cells (Fig. 1C) . Staining was not present in the nucleus and appeared most intense in the peripheral cytoplasm and the cytoplasmic processes of the HTM cells. Serial tangential sections through the meshwork from the inner uveal to the inner cribriform region and inner wall of SC (8–10 sections per specimen, 8–10 µm thick) showed that staining for the enzyme was present in virtually all cells of the uveal, corneoscleral, and cribriform portions of the meshwork.

All control sections incubated without the primary antibody or incubated with a combination of primary antibody and a fivefold excess of tTgase were unstained (Fig. 1D) .

Western Blot of Tissue from the Anterior Eye Segment for tTgase
Western blot analysis for tTgase performed with homogenates of HTM, sclera, iris, ciliary muscle, and ciliary process tissues showed a single band at approximatly 80 kDa. In the scleral tissue there was almost no tTgase, whereas the HTM, ciliary muscle, and ciliary processes contained the protein. Quantification showed the highest amount of tTgase in the iris, moderate and comparable amounts in the HTM and the ciliary muscle, and the lowest amount in the ciliary processes (Fig. 2) .

View larger version (61K): [in this window] [in a new window]   Figure 2. Western blot analysis of tissue transglutaminase (tTgase) in different specimens of the anterior segment of the eye. 1, iris; 2, TM; 3, sclera; 4, ciliary muscle; 5, ciliary process. Lysates from approximately equal amounts of protein (2 µg) were separated by SDS-PAGE and blotted for immunochemical detection of tTgase content. The number below each band is the chemiluminescence measurement. MW, molecular weight.

View larger version (162K): [in this window] [in a new window]   Figure 3. Immunohistochemical staining of tissue transglutaminase in fixed and permeabilized HTM cells (magnification, x160). All cells are intensely stained. The staining is mainly present in the cytoplasm of the cells. Treatment with either 1.0 ng/ml TGF-ß1, 1.0 ng/ml TGF-ß2, or 5 x 10-7 M DEX for 24 hours did not change the staining intensity or staining pattern (data not shown).

View larger version (90K): [in this window] [in a new window]   Figure 4. Confluent HTM cultures stained for extracellular tissue transglutaminase (tTgase; red) and fibronectin (green). (A) In untreated control cells, staining for extracellular tTgase was only sparse. (B) After treatment with 1.0 ng/ml TGF-ß2 for 24 hours, extracellular staining for tTgase was much more prominent than in untreated controls. (C) In untreated controls, staining for fibronectin and tTgase was sparse. Only at places there was colocalization of fibronectin and tTgase (yellow). (D) After treatment with TGF-ß2 the amount of the green-stained, extracellular fibronectin increased markedly, and there were numerous yellow-stained strands visible between the cells, indicating an increase in colocalization of tTgase and fibronectin. Magnification, (A through D) x160.

Northern Blot Analysis of tTgase.
Northern blot analysis of untreated HTM cells showed a single faint band after hybridization with an antisense tTgase RNA probe, which was 3.5 kb in length (Fig. 5A ). Treatment with either TGF-ß1 or TGF-ß2 significantly increased the levels of tTgase mRNA after 12 hours of treatment (Fig. 5A) . Quantification in relation to the methylene blue–stained 28S bands showed a five- to sixfold increase after treatment with either 1.0 ng/ml TGF-ß1 or -ß2. Treatment of HTM cells for 96 hours with TGF-ß1 or -ß2 showed nearly the same results (Fig. 5A) . The quantification showed a four- to sevenfold increase. Treatment for 24 and 48 hours with TGF-ß1 or -ß2 also showed a four- to sevenfold increase (data not shown). DEX treatment for 12, 24, 48, and 96 hours had no effect on tTgase mRNA expression in HTM cells (Fig. 5A ; data for 24 and 48 hours not shown).

View larger version (44K): [in this window] [in a new window]   Figure 5. (A) Northern blot analysis of tissue transglutaminase (tTgase) mRNA in confluent HTM cells 12 and 96 hours after treatment with either 1.0 ng/ml TGF-ß1, -ß2, or 5 x 10-7 M DEX. (B) Methylene blue staining of the 28 and 18S rRNA bands is also shown, demonstrating relative integrity and even loading of the RNA. MW, molecular weight; Co., control; RDI, relative densitometric intensity (normalized to 28 seconds rRNA). (C) Western blot analysis of tTgase in HTM monolayers treated as described for Northern blot analysis. Lysates from approximately equal amounts of protein (2 µg) were separated by SDS-PAGE and blotted for immunochemical detection of tTgase content. The number below each band shows the chemiluminescentce measurement. MW, molecular weight, Co., control. After treatment with TGF-ß1 and -ß2, there is a prominent increase in expression of the mRNA for tTgase (A) as well as of the protein (C). DEX treatment had no effect.

Northern Blot Analysis of Fibronectin.
Hybridization of mRNA from HTM cells with an antisense fibronectin RNA probe (Fig. 6) showed a three- to fourfold increase after 24 hours treatment with TGF-ß compared with that in untreated HTM cells. TGF-ß1 or -ß2 showed essentially similar results; that is, both mediators increased the fibronectin-specific mRNA (7.7 kb in length) to a similar degree.

View larger version (26K): [in this window] [in a new window]   Figure 6. Northern blot analysis of fibronectin mRNA in confluent HTM cells 24 hours after treatment with either 1.0 ng/ml TGF-ß1 or -ß2. Methylene blue staining of the 28 and 18S rRNA bands is also shown, demonstrating relative integrity and even loading of the RNA. MW, molecular weight; Co., control; RDI, relative densitometric intensity (normalized to 28 seconds rRNA). Treatment with TGF-ß1 and -ß2 increased expression of fibronectin mRNA.

View larger version (68K): [in this window] [in a new window]   Figure 7. Fibronectin polymerization was analyzed by plating HTM cells onto uncoated plates. HTM cells were kept confluent for at least 7 days and then treated for 24 hours with either 1.0 ng/ml TGF-ß1 or -ß2. After 24 hours of incubation, cells were solubilized in reducing Laemmli loading buffer. After separation of proteins by SDS-PAGE, the whole gel, including the stacking gel, was Western blotted. The membrane was immunoprobed with an antibody against fibronectin. TSG, top of the stacking gel; TRG, top of the resolving gel; MW, molecular weight, Co., control. After treatment with TGF-ß1 and -ß2, there was a marked increase in both the fibronectin monomere as well as in the polymerized fibronectin seen in the TSG.

View larger version (15K): [in this window] [in a new window]   Figure 8. Cell-mediated incorporation of BTC into fibronectin by tissue tTgase using either untreated (Co.) or treated (TGF-ß1 or -ß2) HTM cells. Treated cells were incubated 24 hours under serum-free conditions in the presence of either 1.0 ng/ml TGF-ß1 or -ß2 before seeding. HTM cells were plated (2 x 104 cells/well) in complete DMEM medium without serum in the presence of 0.1 mM BTC. Cells were allowed to incubate for different time periods (0, 5, 10, 20, 40, 60, 90, and 120 minutes) at 37°C, and reactions were stopped by washing cells with PBS containing 3 mM EDTA. The negative control (Neg.) was complete DMEM medium without serum in the presence of 0.1 mM BTC. Color development was determined by using an ELISA reader set to 450 nm. Data represent mean values ± SEM from nine experiments with three different cell cultures. HTM cells treated 24 hours before seeding with either TGF-ß1 and -ß2 showed a markedly increased incorporation of BTC compared with that of untreated controls.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

tTgases are enzymes catalyzing reactions between glutaminyl residue and different amines, which result in the formation of covalent cross-linking -(-glutamyl) lysine bonds that are resistant to enzymatic degradation.¹⁶ An increase in tTgase activity has been shown in a considerable number of pathologic conditions in which an increase in cross-linked proteins is assumed to be a causative factor. An increase in -(-glutamyl) lysine cross-links of ECM material induced by increase in tTgase activity was observed in paraquat-induced pulmonary fibrosis,⁴⁷ arteriosclerosis,^{48 49 50} neurofibrillary tangles in Alzheimer disease,^{51 52} and in renal fibrosis.⁵³ The factors responsible for induction of tTgase activity are not known. In renal fibrosis, increase in TGF-ß has been discussed as one possible mediator for the observed increase in tTgase.⁵³ In fact, enhanced expression of tTgase by TGF-ß has been reported in rabbit tracheal epithelial cells,⁵⁴ human epidermal keratinocytes,⁵⁵ and in rat hepatoma cell lines.⁵⁶ However, other factors can also induce tTgase expression; for example, in rat hepatoma cell lines, induction of tTgase has been demonstrated after DEX treatment.⁵⁶ Human promyelytic leukemia HL 60 cells⁵⁷ and mouse peritoneal macrophages^{58 59} respond to retinoic acid treatment with induction of tTgase expression and differentiation. Sodium butyrate induces tTgase in human lung fibroblast cells.⁶⁰ Dimethyl sulfoxide and n-butyric acid increase tTgase activity in the Friend erythroleukemia cell line GM979.⁶¹

In the eye, tTgase has been shown in cataractous lenses⁶² and in the retina of Royal College of Surgeons (RCS) rats developing hereditary retinal degeneration and light-induced retinal damage.⁶³ The intracellular lens transglutaminase catalyzes the formation of ß-crystallin dimers by -(-glutamyl) lysine chain bridges.⁶⁴ It was discussed that these cross-links are involved in cataract formation. In RCS rats, increased retinal tTgase activity cross-linked intracellular proteins through the formation of -(-glutamyl) lysine isopeptide bonds in cells undergoing apoptosis.⁶³ Our data show that in the eye, tTgases are also constitutively expressed in a variety of ocular tissues, including the entire TM. Constitutive expression of tTgase has been shown in a variety of tissues.^{23 24 25} The cross-linking action of tTgases seems to be important not only in pathology, but also under normal conditions for purposes of stabilizing structural proteins and ECM–cell interaction.^{22 65 66} We assume that the enzyme serves the same stabilizing function in the eye.

It is well established that in glaucomatous TM, the ECM is significantly increased.² This increase might be due to an increase in cross-linking activity of tTgase, thereby inhibiting ECM degradation by metalloproteinases. As has been discussed before, in other systems tTgase expression can be induced by TGF-ß. Because TGF-ß2 levels are increased in a number of glaucomatous eyes, in this study we investigated whether TGFß treatment of HTM cells might increase tTgase activity and whether tTgase cross-links ECM produced by HTM cells. In normal eyes, the average level of the activated form of TGF-ß2 was approximately 0.15 ng/ml, whereas in POAG eyes it was 0.5 to 2.0 ng/ml.^{33 34} Therefore, we used 1.0 ng/ml TGF-ß2 for treatment of HTM cells. Our finding of an increase in tTgase expression and cross-linking of fibronectin strongly suggests that an increase in tTgase activity plays a role in augmentation of ECM in the TM. Other in vitro studies have reported that tTgase enhances conversion of latent TGF-ß to active TGF-ß.^{67 68 69} If this holds true for the TM, an increase in tTgase expression could establish a vicious circle.

We do not yet know whether TGF-ß2 also increases tTgase expression and activity in vivo. Still, it is tempting to speculate that increased TGF-ß in the aqueous humor of glaucomatous eyes induces expression of extracellular tTgase and thereby quantitative and qualitative changes of the ECM. Such changes may finally lead to augmentation of ECM in the TM and an increase in outflow resistance in glaucoma.

	Acknowledgements

 
The authors thank Angelika Pach, Julia Mausolf, Sandra Hartmann, Barbara Teschemacher, and Marco Gößwein for expert technical assistance.

	Footnotes

 
Supported by SFB 539 (Glaukome) der Deutschen Forschungsgemeinschaft Bonn, Biomed PL 961593 of the European Commission and the Academy of Science, Mainz, Germany.

Submitted for publication April 7, 1999; revised September 28, 1999 and January 31, 2000; accepted February 15, 2000.

Commercial relationships policy: N.

Corresponding author: Elke Lütjen-Drecoll, Department of Anatomy II, University of Erlangen-Nürnberg, Universitätsstraße 19, D-91054 Erlangen, Germany. anat2.gl{at}anatomie.uni-erlangen.de

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