Transforming growth factor-beta 2 modulated extracellular matrix component expression in cultured human optic nerve head astrocytes.
OBJECTIVE
To study whether glaucomatous extracellular matrix (ECM) modifications in the lamina cribrosa might be induced by TGF-beta 2, the effect of TGF-beta 2 on the expression of collagen types I (Col1 alpha 1), III (Col3 alpha 1), and IV (Col4 alpha 2); fibronectin (FN); tissue transglutaminase (TGM2); connective tissue growth factor (CTGF); and thrombospondin (TSP-1) in cultured human optic nerve head (ONH) astrocytes was investigated.


METHODS
Astrocytes were isolated from eyes of five human donors, and cultured monolayers were treated with 1.0 ng/mL TGF-beta 2 for 24 and 48 hours. Expression of Col1 alpha 1, Col3 alpha 1, Col4 alpha 2, FN, TGM2, CTGF, and TSP-1 was examined by semiquantitative RT-PCR and Northern and Western blot analyses. The effect of CTGF silencing on the TGF-beta 2-modulated expression of these genes was investigated by transfection of CTGF small interfering (si)RNA before TGF-beta 2 treatment.


RESULTS
TGF-beta 2 treatment upregulated the expression of Col1 alpha 1, Col4 alpha 2, FN, CTGF, TGM2, and TSP-1 mRNA and protein in cultured astrocytes. Inductions ranged between 1.5- and 4-fold. Expression of Col3 alpha 1 remained unaffected. Transfection of 10 nM CTGF siRNA inhibited the TGF-beta 2-induced upregulation of CTGF, Col4 alpha 2, Col1 alpha 1, TGM2, and FN, whereas TSP-1 expression was not reduced.


CONCLUSIONS
TGF-beta 2 is capable of inducing the expression of ECM and basement membrane components in cultured ONH astrocytes via CTGF and upregulated TSP-1, a protein naturally involved in the activation of latent TGF-beta. Therefore, TGF-beta 2 could be a factor in the initiation of the modification of ECM in the glaucomatous ONH. In addition, TSP-1 induction may be a mechanism by which TGF-beta 2 amplifies its own activation.
Fuchshofer
Rudolf
R
Department of Anatomy II, Friedrich Alexander University, Erlangen, Germany.
Birke
Marco
M
Welge-Lussen
Ulrich
U
Kook
Daniel
D
Lütjen-Drecoll
Elke
E
D000368:Aged; D000369:Aged, 80 and over; D001253:Astrocytes / Q000187:drug effects* / Q000378:metabolism; D015152:Blotting, Northern; D015153:Blotting, Western; D002478:Cells, Cultured; D003094:Collagen / Q000235:genetics / Q000378:metabolism; D055513:Connective Tissue Growth Factor; D016326:Extracellular Matrix Proteins / Q000235:genetics* / Q000378:metabolism; D005353:Fibronectins / Q000235:genetics / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D017874:Immediate-Early Proteins / Q000235:genetics / Q000378:metabolism; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D008875:Middle Aged; D009898:Optic Disk / Q000187:drug effects* / Q000378:metabolism; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019700:Thrombospondin 1 / Q000235:genetics / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D053781:Transforming Growth Factor beta2; D011503:Transglutaminases / Q000235:genetics / Q000378:metabolism; D015854:Up-Regulation
C526317:CCN2 protein, human; D016326:Extracellular Matrix Proteins; D005353:Fibronectins; D017874:Immediate-Early Proteins; D036341:Intercellular Signaling Peptides and Proteins; D012333:RNA, Messenger; C509041:TGFB2 protein, human; C000717608:TGM2 protein, human; D019700:Thrombospondin 1; D016212:Transforming Growth Factor beta; D053781:Transforming Growth Factor beta2; D055513:Connective Tissue Growth Factor; D003094:Collagen; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011503:Transglutaminases; D019204:GTP-Binding Proteins