The effect of TGF-beta2 on elastin, type VI collagen, and components of the proteolytic degradation system in human optic nerve astrocytes.
OBJECTIVE
To investigate the effect of transforming growth factor (TGF)-beta2 on the expression of (1) elastin and type VI collagen (ColVI), (2) extracellular matrix (ECM)-degrading matrix-metalloproteinases (MMPs) and regulators of their activity/activation, and (3) the involvement of connective tissue growth factor (CTGF) in the TGF-beta2-mediated regulations in cultured human type-1A and -1B optic nerve astrocytes.


METHODS
Astrocytes were isolated from the optic nerves of 11 donors aged 19 to 62 years without a history of eye disease from the prelaminar (type 1B, five explants) or postlaminar (type 1A, six explants) region. Cultures of passages 3 to 5 were treated with 1 ng/mL recombinant human TGF-beta2 for 72 hours, and regulatory effects on the expression of elastin and the ColVI chains alpha1, alpha2, and alpha3; MMP-1, -2, -3, -7, -9, -12, and -13; tissue inhibitors of MMPs (TIMPs) -1, -2, and -3; plasminogen activator inhibitor 1 (PAI-1); and urokinase and tissue plasminogen activators (uPA, tPA) were initially analyzed by RT-PCR and confirmed and quantified by real-time PCR (rtPCR). The regulation of proteins was studied by Western blot analysis, and MMP-2 activity was assessed by gelatin zymography. The involvement of CTGF was tested by knockdown experiments with CTGF-small interfering (si)RNA.


RESULTS
TGF-beta2 increased the expression of elastin (5X[rtPCR]/6X[WB]), ColVIalpha2 (3X/5X), ColVIalpha3 (7X/9X), MMP-2 (2X/2X), TIMP-1/-3 (1.5X/2X), and PAI-1 (8X/4X) compared to untreated controls. tPA was reduced to 0.5X. MMP-1, -3, -7, and -12 and TIMP-2 were expressed but were not responsive to TGF-beta2. MMP-9 and -13 and uPA were marginally expressed and close to the detection threshold. MMP-2 activity was significantly reduced in gelatin zymography. Transfection of CTGF-siRNA blocked TGF-beta2-mediated activation of elastin and ColVI but had no effect on MMP-2 and PAI-1 induction. Type 1A and 1B astrocytes reacted identically.


CONCLUSIONS
TGF-beta2 induces expression of elastin and ColVI and thereby could contribute to the increase of type VI collagen fibers in the tissue septae and the elastotic changes typically observed in POAG. With the concurrent activation of TIMP-1 and -3 and PAI-1 and the repression of tPA, TGF-beta2 could negatively regulate the activity and activation of MMPs. This effect could further amplify ECM accumulation and elastosis.
Neumann
Carolin
C
Department of Anatomy II, Friedrich Alexander University, Erlangen, Germany.
Yu
Alice
A
Welge-Lüssen
Ulrich
U
Lütjen-Drecoll
Elke
E
Birke
Marco
M
D000328:Adult; D001253:Astrocytes / Q000187:drug effects* / Q000378:metabolism; D015153:Blotting, Western; D002478:Cells, Cultured; D024142:Collagen Type VI / Q000378:metabolism*; D055513:Connective Tissue Growth Factor; D004549:Elastin / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D017874:Immediate-Early Proteins / Q000235:genetics / Q000378:metabolism; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D020782:Matrix Metalloproteinases / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008875:Middle Aged; D009900:Optic Nerve / Q000166:cytology*; D034741:RNA, Small Interfering / Q000378:metabolism; D011994:Recombinant Proteins / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019714:Tissue Inhibitor of Metalloproteinases / Q000378:metabolism*; D053781:Transforming Growth Factor beta2 / Q000494:pharmacology*
C526317:CCN2 protein, human; D024142:Collagen Type VI; D017874:Immediate-Early Proteins; D036341:Intercellular Signaling Peptides and Proteins; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D019714:Tissue Inhibitor of Metalloproteinases; D053781:Transforming Growth Factor beta2; D055513:Connective Tissue Growth Factor; D004549:Elastin; D020782:Matrix Metalloproteinases