Abstract
Purpose: :
TGFβ2 is elevated in the aqueous humor of glaucoma patients and increases extracellular matrix (ECM) deposition in the trabecular meshwork (TM) as well as increases intraocular pressure in perfusion cultured anterior segments and rodent eyes. TGFβ2 increases the expression and activity of TGM2, which cross-links ECM molecules making them more resistant to degradation. We have investigated the signaling pathway by which TGFβ2 induces TGM2 in human TM cells.
Methods: :
Immunohistochemistry was used to evaluate the expression and co-localization of TGFβ2 and TGM2 in 3 normal and 3 glaucomatous TM tissues. Three normal human TM cell lines and 3 glaucomatous TM cell lines were evaluated for endogenous TGFβ2 levels by western immunoblot analysis. These TM cell lines were also treated with or without TGFβ2 (5 ng/ml) in serum-free medium for 48 hrs. Activation of Smad 2/3, p38, and ERK signaling pathways was evaluated by western immunoblot analysis using phospho-specific antibodies. siRNAs were used to suppress Smad2, Smad3, p38, ERK1/2, and CTGF expression.
Results: :
Immunohistochemistry studies indicated elevated expression and co-localization of both TGFβ2 and TGM2 in glaucoma TM tissues. Endogenous TGFβ2 levels were also significantly elevated in cultured GTM cells (p<0.05). TGFβ2 treatment increased the phosphorylation of Smad3, p38 and ERK1/2 in all six TM cell lines. Although p38, ERK1/2, and CTGF siRNAs selectively knocked down target protein expression (p<0.05), this did not prevent TGFβ2 induction of TGM2. In contrast, siRNA knock-down of Smad 2 or Smad3 (p<0.05) suppressed TGFβ2 induction of TGM2.
Conclusions: :
There was increased expression and co-localization of TGFβ2 and TGM2 in glaucomatous TM tissues and elevated endogenous TGFβ2 levels in GTM cells. TGFβ2 treatment of cultured TM cells activated both the canonical Smad and non-Smad pathways. However, TGFβ2 induction of TGM2 was mediated via the Smad pathway and did not appear to involve CTGF. Regulation of the Smad signaling pathway in the TM may be useful in the therapy of glaucoma associated with aberrant TGFβ2 signaling.
Keywords: trabecular meshwork • growth factors/growth factor receptors • extracellular matrix