Smad3 is necessary for transforming growth factor-beta2 induced ocular hypertension in mice

TGF beta 2 induces extracellular matrix (ECM) remodeling and alters the cytoskeleton by both the canonical Smad and non-canonical signaling pathways. TGF beta 2 regulates the expression of ECM proteins in trabecular meshwork (TM) cells, increases intraocular pressure (IOP) in an ex vivo perfusion organ culture model, and induces ocular hypertension in rodent eyes. A necessary step in the canonical Smad signaling pathway is phosphorylation of receptor protein Smad3 by the TGF-beta receptor complex. The purpose of this study was to determine whether TGF beta 2 signals in vivo through the canonical Smad signaling pathway in the TM using Smad3 knockout (KO) mice. Ad5.hTGF-beta(226/228) (2.5 x 10(7) pfu) was injected intravitreally into one eye of homozygous (WT), heterozygous (HET), and homozygous (KO) 129-Smad3(tm1Par)/J mice (n = 9-10 mice/group), with the uninjected contralateral eye serving as the control. IOP measurements were taken using a rebound tonometer. To test the effect of TGF beta 2 signaling on the ECM, fibronectin expression was determined by immunohistochemistry and qPCR analysis. Transduction of the TM with viral vector Ad5.hTGF beta 2(226/228) caused a statistically significant difference in IOP exposure between Smad3 genotypes: WT, 187.7 +/- 23.9 mmHg*day (n = 9); HET, 95.6 +/- 24.5 mmHg*day (n = 9); KO, 52.8 +/- 25.2 mmHg*day (n = 10); (p < 0.05 WT versus HET, p < 0.01 WT versus KO). Immunohistochemistry and qPCR analysis showed that Ad5.hTGF beta 2(226/228) increased fibronectin expression in the TM of WT mice (2.23 +/- 0.24 fold) compared to Smad3 KO mice (0.99 +/- 0.19 fold), p < 0.05. These results demonstrate Smad3 is a necessary signaling protein for TGF beta 2-induced ocular hypertension and fibronectin deposition in the TM. (C) 2013 Elsevier Ltd. All rights reserved.