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Alexandra Goulart Castillejos, Min Hyung Kang, Douglas J Rhee; Suppression of Parvin-a in Human Trabecular Meshwork Downregulates Extracellular Matrix Components. Invest. Ophthalmol. Vis. Sci. 2017;58(8):3493.
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© ARVO (1962-2015); The Authors (2016-present)
Secreted Protein Acidic in Cysteine (SPARC), a matricellular protein, has been shown to regulate the homeostasis of the extracellular matrix (ECM) of the trabecular meshwork (TM) of the eye.1 This is of particular interest in the pathogenesis of primary open angle glaucoma where intraocular pressure increases due to decreased drainage of aqueous humor out the TM. Our purpose is to elucidate the mechanistic pathway(s) SPARC utilizes to increase the levels of certain ECM proteins. Integrin-linked kinase (ILK) is a receptor for SPARC.2 ILK forms a complex with PINCH and parvin called the ‘PIP complex’.3 We hypothesized that parvin-a is critical to SPARC’s downstream signaling at that suppressing parvin-a will lead to a decreased ECM levels.
A lentivirus carrying the short hairpin RNA targeting human parvin-a (shparvin-a) was constructed using the pLKO.1 vector system. shparvin-a was then used to suppress parvin-a in primary cultures of human TM endothelial cells using an MOI of 10. Selected ECM proteins from cell lysates and conditioned media were analyzed by immunoblotting.
Immunoblot analysis showed that treatment of lenti-shparvin-a suppressed the expression of parvin-a as compared to shcontrol in TM cells. Cells with suppressed parvin-a showed significantly attenuated levels of Fibronectin (n=5, p=0.0154), Collagen I (n=3, p=0.0240), Collagen VI (n=4, p=0.0135) and Laminin (n=4, p=0.0293). SPARC (n=3, p=0.4784), AP1 (n=2, p=0.9284) and Rac1 (n=2, p=0.6608) levels were unchanged by parvin-a when compared with shcontrol.
Suppression of parvin-a causes a decrease in ECM proteins such as Collagen IV, Collagen I, Collagen VI, Fibronectin and Laminin, suggesting that parvin-a may play a regulatory role in the downstream signaling of SPARK-ILK.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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