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A. F. Clark, G. S. Zode, A. Sethi, R. J. Wordinger; TGFβ2 Increases Extracellular Matrix Proteins in Human Optic Nerve Head Cells via Activation of the Smad Signaling Pathway. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4318.
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© ARVO (1962-2015); The Authors (2016-present)
TGFβ2 appears to play a role in the pathogenic remodeling of the glaucomatous optic nerve head (ONH). We wanted to determine which of the several signaling pathways is involved in TGFβ2 induction of extracellular matrix (ECM) proteins in human ONH cells.
Normal (n=4) and glaucomatous (n=4) donor eyes were examined by immunohistochemistry for TGFβ2 expression in the ONH. Cultured human ONH astrocytes (ONHAs) and lamina cribrosa (LC) cell lines (n=3 each) were treated with or without TGFβ2 in the presence or absence of a TGFβR1 inhibitor (SB431542), and inhibitor of Smad 3 phosphorylation (SIS3), or siRNAs targeting Smad2 or Smad3. Effects on fibronectin, collagen, PAI-1, and elastin expression in cell lysates and conditioned medium were determined by western immunoblot and ELISA analyses. Activation of Smad, p38, ERK1/2, and JNK1/2 was determined by western blot analysis.
TGFβ2 expression was significantly higher in glaucomatous ONH tissues (p=0.0013) and was associated with GFAP positive cells. Both ONHAs and LC cells made and secreted endogenous TGFβ2. TGFβ2 stimulated the phosphorylation of Smad2 and Smad3, but did not phosphorylate p38, ERK1/2, or JNK1/2. TGFβ2 treatment also increased the co-localization of Smad2/3 with co-Smad4 in the nucleus of both cell types. TGFβ2 significantly increased the expression of fibronectin (p<0.0001), PAI-1, collagens 1 & VI, and elastin. Treatment with SB431542 blocked the TGFβ2 induction of fibronectin (p<0.05), PAI-1, and the phosphorylation of Smads2/3. TGFβ2 induction of fibronectin and phosphorylation of Smad3 was blocked by SIS3. Treatment with either Smad2 or Smad3 siRNAs selectively knocked down Smad2/3 expression (p<0.002) and inhibited TGFβ2 induction of fibronectin (p<0.005) and PAI-1 (p<0.002).
TGFβ2 expression was higher in the glaucomatous ONH. TGFβ2 increased ECM expression in ONHAs and LC cells via the canonical Smad signaling pathway. Select inhibitors of this pathway may prevent glaucomatous remodeling of the ONH ECM.
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