The Effect of TGF-β2 on Elastin, Type VI Collagen, and Components of the Proteolytic Degradation System in Human Optic Nerve Astrocytes

Abstract

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. 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. 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. 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.