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Arumugam Ramachandran Muralidharan, Vasanth Rao; Role of Calponin in Regulating Contractile Activity of Trabecular Meshwork Cells. Invest. Ophthalmol. Vis. Sci. 2017;58(8):3485.
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© ARVO (1962-2015); The Authors (2016-present)
Hypercontractility of the trabecular meshwork (TM) has been demonstrated to influence conventional aqueous humor (AH) outflow and intraocular pressure (IOP). Hence, it is important that we explore and thoroughly understand the regulation of TM contractile activity in the context of glaucoma pathobiology. Towards this objective, in this study we investigated the role of calponin (CNN), an actin-binding, smooth muscle abundant contractile protein possessing myosin II ATPase inhibitory activity, in TM cells and the AH outflow pathway.
The relative expression profile of CNN isoforms (1, 2 & 3) in human- primary TM cells and TM tissue were determined by quantitative RT-PCR analysis and immunoblot analysis. Distribution of CNN 1 and 3 and their co-localization with actin stress fibers in TM cells were analyzed by immunofluorescence and confocal imaging. Regulation of protein levels and activation status (phosphorylation) of CNN 1 and 3 in TM cells by various physiological and pharmacological agents was determined by quantitative immunoblot analysis.
Both TM cells and tissue were found to express all three isoforms of CNN based on qRT-PCR analysis. Consistent with the expression pattern, immunoblot analysis also confirmed the presence of CNN 1 and 3 in TM cells. Immunofluorescence analysis revealed co-localization of CNN (isoform 1&3) with actin stress fibers in TM cells. Serum starved TM cells treated with lysophosphatidic acid (5 μM/6 hr), TGF-β2 (10ng/ml, 24 hr), dexamethasone (0.5μM/96 hr), angiotensin II (1μM/24 hr) and endothelin-1 (2μM/24 hr) showed a significant increase in both CNN 1 and 3 protein levels compared to controls. Similarly, the levels of phosphorylated CNN 3 were increased in TM cells under the above described treatment conditions. Pharmacological inhibition of Rho kinase and protein kinase C using Y27632 (10μM/18 hr) and GF109203X (20μM/6 hr), respectively, led to significant decreases in the levels of phosphorylated CNN 3 in TM cells.
Taken together, these results reveal that both the expression and phosphorylation-status of CNN which are known to influence the contractile activity of smooth muscle, are responsive to various external factors and intracellular signaling molecules in TM cells. This study also identifies CNN as one of the molecular targets for Rho kinase inhibitors in TM cells and its potential role in AH outflow and homeostasis of IOP.
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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