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Michael Lucio De Ieso, Megan Kuhn, Michael H Elliott, Daniel W Stamer; Caveolin-1 mediates relaxation of trabecular meshwork cells to chronic mechanical stretch via protein kinase C. Invest. Ophthalmol. Vis. Sci. 2021;62(8):467.
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Polymorphisms at the Cav1/2 gene loci impart increased risk for ocular hypertension and primary open-angle glaucoma (POAG). Caveolae are specialized cellular domains that form invaginations in the plasma membrane, and Cav1 is required for caveolae biosynthesis. The mechanism by which Cav1 contributes to intraocular pressure (IOP) homeostasis is unknown. Interestingly, protein kinase C (PKC) interacts with Cav1 and plays a key role in trabecular meshwork (TM) contractility; an important mediator of conventional outflow resistance, and subsequently IOP. Thus, we used pharmacological modulators of PKC to test the hypothesis that caveolae serve as mechanosensors in the TM, which respond to changes in IOP by modulating PKC signaling.
Experiments were conducted using cultures of 8 different human TM cell strains. Adenoviruses encoding shRNA targeted to Cav1 were used to silence expression. Western blotting was used to determine relative protein levels, phosphorylation status, and PKC activity. Gö6983 (1 micromolar) was used as a selective PKC inhibitor. For cyclic stretch experiments, TM cells were plated on type IV collagen-coated flexible silicone bottom plates and subjected to 20% stretch, 1 Hz for 24 h. Data are expressed as mean ± SEM.
Using phosphorylated myosin light chain (pMLC) as a surrogate indicator for Rho/ROCK activity and contractile tone, we found that pMLC/MLC levels in TM cells were reduced in stretched verses unstretched conditions (58.2±12% vs. 100±19%, n=8, p=0.005). When Cav1 expression was decreased by knockdown (efficiency=56±5.7%), pMLC/MLC levels were unaffected in stretched verses unstretched conditions (81.5±16% vs. 100±19%, n=8, p=0.28). Levels of pMLC/MLC were also lower in TM cells treated with the PKC inhibitor, Gö6983, for 24 h compared to control (74.1±5.6% vs. 100%, n=7, p<0.004). Interestingly, PKC activity trended downward in 24 h stretched, Cav1-competent TM cells (90.3±9.5% vs. 100±9.3%, n=7, p=0.057). This effect was not present in Cav1-deficient cells (94.5±4.8% vs. 100±9.7%, n=7, p=0.55), likely because PKC colocalizes with Cav1 at plasma membrane in TM cells.
Caveolae act as scaffolds that compartmentalize PKC at the membrane in TM cells, enabling signal transduction to downstream effectors in response to chronic stretch (Figure 1).
This is a 2021 ARVO Annual Meeting abstract.
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