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Jaeyoung Heo, Emily Ahadizadeh, Min Hyung Kang, Yuxi Zheng, Douglas J Rhee; TGF-β2 upregulates the extracellular proteins through SPARC and Integrin-Linked Kinase in Human Trabecular Meshwork. Invest. Ophthalmol. Vis. Sci. 2018;59(9):3529.
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© ARVO (1962-2015); The Authors (2016-present)
Intraocular pressure (IOP) is a known modifiable risk factor for primary open-angle glaucoma (POAG).  Previous studies have shown that transforming growth factor-beta-2 (TGF-β2) and Secreted Protein, Acidic, and Rich in Cysteine (SPARC) play a regulatory role in IOP and extracellular matrix (ECM) composition in the eye. [2,3] TGF-β2 has been shown to upregulate SPARC and ECM proteins such as collagens and fibronectin, which elevate IOP, while decreasing metalloproteases (MMPs) in the trabecular meshwork (TM). [4,5] Integrin-linked kinase (ILK) is a widely expressed serine/threonine kinase that regulates ECM organization through signaling cascades by interacting with SPARC.  We hypothesize that TGF-β2 upregulates ECM proteins through SPARC and ILK in TM.
Human TM cells were treated with adenovirus carrying cDNA of TGF-β2 (Ad-TGF-β2) and a Lentivirus carrying short-hairpin RNA targeting human ILK (Lenti-shILK). The Ad5 vector system was used to construct the adenovirus (Ad-SPARC) and the pLKO.1 vector system was used to construct the Lentivirus (Lenti-shILK). Ad-TGF-β2 was used to upregulate SPARC in the human TM cells at the multiplicity of infection (MOI) 50, and Lenti-shILK was used to inhibit ILK at MOI 5. ECM proteins were analyzed from cell lysates and conditioned media by immunoblotting.
We observed that the overexpression of TGF-β2 by Ad-TGFβ2 upregulated SPARC (1.29±0.56, p=0.20), collagen IV (1.29±0.17, p=0.02) and fibronectin (5.56±4.24, p=0.04) in TM cells, compared by Ad-control. The Lenti-shILK suppressed ILK (0.19±0.11, p=0.16) and mitigated the effect of TGF-β2 on collagen IV (0.42±0.14, p=0.38) and fibronectin (0.53±0.27, p=0.06).
The effect of TGF-β2 on collagen IV and fibronectin are through the SPARC/ILK signaling.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
Figure: Western blot results from Trabecular Meshwork cultures treated with Ad-TGFB2 and Lenti-shILK.
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