Microtubule Disassembly Induces Barrier Dysfunction in Corneal Endothelial Cells

Abstract

Abstract: : Purpose: In response to hypoxia and aging, corneal endothelial (CE) cells manifest variable cell size and shape, referred to as polymegathism and pleomorphism, respectively. These morphological changes herald altered cytoskeleton whose effect on the functional competence of CE in maintaining stromal hydration remains incompletely understood. Our recent studies have shown that disruption of the cortical actin assembly, mobilized through phosphorylation of the regulatory light chain of myosin II (MLC), breaks down barrier integrity of CE. This study extends our observations to microtubules (MT) and is focused on the significance of its disruption on the barrier function of CE. Methods: Experiments were performed with cultured bovine CE (BCEC). Nocodazole (2 µM; ND) was employed to disrupt MT. Since MT disruption is known to activate Rho kinase, we examined its downstream effects including MLC phosphorylation and remodeling of the actin cytoskeleton. MLC phosphorylation was assayed by urea glycerol gel electrophoresis followed by Western blotting. The barrier integrity was assessed as permeability to horseradish peroxidase (HRP). Results:Exposure to ND for 30 min led to significant MLC phosphorylation (∼ 120% of control) as well as several fold increase in the permeability to HRP. The increase in MLC phosphorylation by ND was suppressed by co–treatment with forskolin (10 µM), adenosine (200 µM), or ATP (100 µM), agents which are known to elevate cAMP and suppress thrombin–induced MLC phosphorylation in BCEC. ND also led to disruption of a dense assembly of cortical actin characteristically found in BCEC. TNF–α (100 ng/ml), a pro–inflammatory cytokine known to breakdown barrier dysfunction in CE, also led to MLC phosphorylation (∼ 140% of control) when exposed for 6 hrs. Conclusions: Disruption of MT induces MLC phosphorylation and barrier dysfunction in CE. Since activation of Rho kinase is inhibited by cAMP, the observed reduction of ND–induced MLC phosphorylation suggests mobilization of Rho kinase in response to MT disassembly. TNF–α–induced MLC phosphorylation is also consistent with its ability to cause MT disassembly.

Keywords: cornea: endothelium • cytoskeleton • signal transduction