Tissue transglutaminase as a modifying enzyme of the extracellular matrix in PVR membranes.
OBJECTIVE
Proliferative vitreoretinopathy (PVR) is characterized by the development of epi- and subretinal fibrocellular membranes containing modified retinal pigment epithelial (RPE) cells among others. In the present study, the role of transglutaminases in accumulation of extracellular matrix (ECM) proteins in these membranes was investigated. Transglutaminases are enzymes capable of cross-linking ECM proteins to proteolysis-resistant complexes.


METHODS
PVR membranes were incubated with dansyl-cadaverine to demonstrate active transglutaminase. Localization of tissue transglutaminase (tTgase), its reaction product epsilon-(gamma-glutamyl)-lysine, and fibronectin was investigated immunohistochemically. Colocalization was studied with a confocal laser scanning microscope. PVR membranes were also analyzed by RT-PCR for the presence of tTgase mRNA. In vitro, RPE cells were treated with transforming growth factor-beta2 (TGF-beta2), basic fibroblast growth factor, interleukin-6, and interleukin-1beta. Their effect was studied using immunohistochemistry and Northern and Western blot analyses.


RESULTS
Transglutaminase activity and expression of tTgase were present in all PVR membranes. Staining was most prominent at the rim of the membranes. The enzyme was colocalized with epsilon-(gamma-glutamyl)-lysine and fibronectin. No staining differences were found between epi- and subretinal membranes. Although native RPE cells contained only a basal level of tTgase mRNA, the expression and activity of tTgase was increased under culture conditions and further stimulated by TGF-beta2 treatment.


CONCLUSIONS
The findings demonstrate that in PVR membranes tTgase is present and functionally active. The amount and activity of this enzyme appears to be related to the differentiation state of the RPE cells and their stimulation by TGF-beta2, a growth factor known to be increased in the vitreous of PVR. Intervention at this pathway may open a new approach for PVR prevention and therapy.
Priglinger
Siegfried G
SG
Department of Ophthalmology, Ludwig-Maximilians-University, Mathildenstrasse 8, 80336 Munich, Germany.
May
Christian A
CA
Neubauer
Aljoscha S
AS
Alge
Claudia S
CS
Schönfeld
Carl-L
CL
Kampik
Anselm
A
Welge-Lussen
Ulrich
U
D000293:Adolescent; D000328:Adult; D000368:Aged; D015152:Blotting, Northern; D015153:Blotting, Western; D018929:Cell Culture Techniques; D004151:Dipeptides / Q000378:metabolism; D005109:Extracellular Matrix / Q000201:enzymology*; D016326:Extracellular Matrix Proteins / Q000378:metabolism; D005353:Fibronectins / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D018613:Microscopy, Confocal; D008875:Middle Aged; D010857:Pigment Epithelium of Eye / Q000201:enzymology; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D012333:RNA, Messenger / Q000378:metabolism; D012160:Retina / Q000201:enzymology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D011503:Transglutaminases / Q000235:genetics / Q000378:metabolism*; D018630:Vitreoretinopathy, Proliferative / Q000201:enzymology*
D004151:Dipeptides; D016326:Extracellular Matrix Proteins; D005353:Fibronectins; D012333:RNA, Messenger; C001299:epsilon-(gamma-glutamyl)-lysine; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011503:Transglutaminases; D019204:GTP-Binding Proteins