TGF-beta2-induced cell surface tissue transglutaminase increases adhesion and migration of RPE cells on fibronectin through the gelatin-binding domain.
OBJECTIVE
Migration and adhesion of dislocated retinal pigment epithelial (RPE) cells to a fibronectin-rich extracellular matrix is an initial step in proliferative vitreoretinopathy (PVR). In the present study, the functional role of cell surface tissue transglutaminase (tTG) in adhesion and migration of RPE cells on fibronectin (Fn) and collagen type I (Col I) after stimulation with TGF-beta2 was investigated.


METHODS
Cultured human RPE cells were treated with 1.0 ng/mL TGF-beta2 for 24 hours. Cell surface tTG expression was determined by cell fraction analysis. Attachment on Col I, full-length Fn, and its 45-kDa gelatin-binding and 110-kDa cell-binding fragment was measured with an MTT assay. Migration of RPE cells was measured by a Boyden chamber assay, and cell spreading was determined. Experiments were performed in the presence or absence of anti-tTG antibodies and anti-integrin alpha5 and beta1 antibodies.


RESULTS
TGF-beta2 markedly induced expression of cell-surface tTG on RPE cells and increased attachment and migration on Fn and Col I. Blocking cell surface tTG inhibited attachment, migration, and spreading on Fn and its 45-kDa gelatin-binding fragment, whereas no effect was seen on Col I and the 110-kDa cell-binding Fn fragment. In contrast, blocking of integrin alpha5 and beta1 suppressed adhesion and migration on full-length Fn and the 110-kDa Fn fragment.


CONCLUSIONS
These data demonstrate that TGF-beta2 increases expression of cell surface tTG, which in turn strengthens adhesion, migration, and spreading of RPE cells on Fn through the 45-kDa gelatin-binding Fn fragment. At the onset of PVR, this mechanism may help RPE cells to attach and migrate on Fn-containing matrices.
Department of Ophthalmology, Ludwig-Maximilians-University, Munich, Germany.
Siegfried G
SG
Priglinger
Claudia S
CS
Alge
Aljoscha S
AS
Neubauer
Nadine
N
Kristin
Christoph
C
Hirneiss
Kirsten
K
Eibl
Anselm
A
Kampik
Ulrich
U
Welge-Lussen
D000293:Adolescent; D000328:Adult; D000368:Aged; D001665:Binding Sites; D015153:Blotting, Western; D002448:Cell Adhesion / Q000502:physiology; D002462:Cell Membrane / Q000187:drug effects; D002465:Cell Movement / Q000502:physiology; D002478:Cells, Cultured; D024042:Collagen Type I / Q000378:metabolism; D005353:Fibronectins / Q000378:metabolism*; D005780:Gelatin / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism; D008875:Middle Aged; D010857:Pigment Epithelium of Eye / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology*; D011485:Protein Binding; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D053781:Transforming Growth Factor beta2; D011503:Transglutaminases / Q000378:metabolism*
D024042:Collagen Type I; D005353:Fibronectins; D008565:Membrane Proteins; C509041:TGFB2 protein, human; D016212:Transforming Growth Factor beta; D053781:Transforming Growth Factor beta2; D005780:Gelatin; D011503:Transglutaminases