title: Smad3 is necessary for transforming growth factor-beta2 induced ocular hypertension in mice.
abstract: TGFβ2 induces extracellular matrix (ECM) remodeling and alters the cytoskeleton by both the canonical Smad and non-canonical signaling pathways. TGFβ2 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-β receptor complex. The purpose of this study was to determine whether TGFβ2 signals in vivo through the canonical Smad signaling pathway in the TM using Smad3 knockout (KO) mice. Ad5.hTGFβ2(226/228) (2.5 × 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 TGFβ2 signaling on the ECM, fibronectin expression was determined by immunohistochemistry and qPCR analysis. Transduction of the TM with viral vector Ad5.hTGFβ2(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.hTGFβ2(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 TGFβ2-induced ocular hypertension and fibronectin deposition in the TM.
authors.affiliation: North Texas Eye Research Institute, Department of Cell Biology and Anatomy, University of North Texas Health Science Center, CBH-441, 3500 Camp Bowie Blvd, Fort Worth, TX 76107, USA.
authors.forename: Colleen M
authors.initials: CM
authors.lastname: McDowell
authors.forename: Holly E
authors.initials: HE
authors.lastname: Tebow
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Wordinger
authors.forename: Abbot F
authors.initials: AF
authors.lastname: Clark
mesh_terms: D000818:Animals; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D051379:Mice; D009798:Ocular Hypertension / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D015398:Signal Transduction; D051900:Smad3 Protein / Q000235:genetics* / Q000378:metabolism; D014129:Trabecular Meshwork / Q000378:metabolism*; D053781:Transforming Growth Factor beta2 / Q000235:genetics* / Q000378:metabolism
chemical_list: D012333:RNA, Messenger; D051900:Smad3 Protein; D053781:Transforming Growth Factor beta2
keywords: TGFβ2 signaling; glaucoma; mouse model; ocular hypertension; trabecular meshwork