Thrombin-induced phosphorylation of the regulatory light chain of myosin II in cultured bovine corneal endothelial cells
Introduction
Corneal transparency is dependent on the hydration of corneal stroma, which is held constant by the corneal endothelium (CE). Leakage from the aqueous humor into the stroma is a principal threat to stromal hydration. It is induced by the imbibition pressure associated with stromal glycosaminoglycans. This fluid leak, determined by the barrier integrity of CE, is counterbalanced by active fluid transport mechanisms expressed in CE (Riley et al., 1998, Bonanno, 2003).
In general, the barrier integrity of cellular monolayers is localized at tight junctions. Trans-membrane proteins associated with the tight junctions of two neighbouring cells interact with one another, occluding the paracellular space. This interaction is influenced by the contractility of the cortical actin cytoskeleton among other factors. In vascular endothelium and in certain epithelia, the cortical actin cytoskeleton forms a dense band at the periphery (perijunctional actomyosin ring or PAMR), which provides the tethering forces necessary to maintain apposition between neighbouring cells at tight and adherens junctions (Turner et al., 1999, Turner, 2000). An increase in the contractility of PAMR brings about a centripetal force that opposes the tethering forces. Such a phenomenon is known to cause a breakdown in barrier integrity (Garcia et al., 1995a, Turner et al., 1997, Turner et al., 1999, Stevens et al., 2000, Turner, 2000, Wang et al., 2001).
The contractility of the actin cytoskeleton, including that of PAMR, is regulated through acto-myosin interaction that is induced by the phosphorylation of the regulatory light chain of myosin II, also called myosin light chain (MLC; MW 20 kDa) (Somlyo and Somlyo, 2000, Kamm and Stull, 2001). Myosin light chain kinase (MLCK) is a dedicated protein kinase that catalyses MLC phosphorylation at Ser-19 and Thr-18 residues. The affinity of MLCK to MLC is enhanced when the enzyme is bound to the Ca2+-calmodulin complex (Somlyo and Somlyo, 2000, Kamm and Stull, 2001). This brings about Ca2+- sensitive MLC phosphorylation and a consequent increase in actin contractility in smooth muscle and non-smooth muscle cells. In opposition to the activity of MLCK, MLC phosphatase (MLCP) dephosphorylates MLC. The activity of MLCP is inhibited by the phosphorylation of its putative regulatory subunit. Thus, PKC and Rho kinase-1 (Rho-associated coiled coil-containing protein kinase-1 or ROCK-1; effector of RhoA), which are known to phosphorylate MLCP, induce an increase in contractility of the actin cytoskeleton in a Ca2+-insensitive manner (Somlyo and Somlyo, 2000, Kamm and Stull, 2001). PKA, on the other hand, phosphorylates MLCK, leading to its inactivation and causing relaxation of the actin cytoskeleton (Garcia et al., 1997, Somlyo and Somlyo, 2000, Kamm and Stull, 2001). These observations suggest that G-protein coupled receptors (GPCRs) signalling through their effectors such as PKA, PKC, or Rho kinase-1 (Sah et al., 2000, Somlyo and Somlyo, 2000), influence MLC phosphorylation and result in altered contractility of the actin cytoskeleton. Thus, thrombin, coupled to the activation of and G-proteins, induces rapid MLC phosphorylation and extensive cytoskeletal reorganization in the vascular endothelium. In addition to these changes, a significant breakdown of barrier integrity and the formation of inter-endothelial gaps have been reported (Garcia and Schaphorst, 1995, Garcia et al., 1995a, Vouret-Craviari et al., 1998, Zhao and Davis, 1999, van Nieuw Amerongen et al., 2000, van Hinsbergh and van Nieuw Amerongen, 2002, Vouret-Craviari et al., 2002).
The main objective of this study is to make use of thrombin to ‘screen’ the signal transduction pathways surrounding MLC phosphorylation in CE to better understand the role of the cytoskeleton and its effects on barrier integrity in stromal hydration control. Previous studies, which examined the thrombin-induced response in CE, focused on the role of PKC (Sakamoto et al., 1995). This study is unique in that it shows the existence of Rho kinase-1-dependent signalling pathways in CE operating in addition to PKC in the thrombin-mediated cytoskeleton reorganization and breakdown of barrier integrity.
Section snippets
Cell culture
Primary cultures of bovine CE cells (BCEC) from fresh eyes were established in Dulbecco's Modified Eagle's Medium (DMEM), supplemented with 10% foetal calf serum and an antibiotic–antimycotic mixture (Penicillin 100 U ml−1, Streptomycin 100 μg/ml and Fungizone 0·25 μg/ml) (Srinivas et al., 2002). Cells were cultured at 37 °C in a humidified atmosphere containing 5% CO2 and 95% air while being fed at 48-hr intervals. Second and third passage cultures, grown to confluence on glass coverslips or Petri
Expression of MLCK, PAR-1, RhoA, and Rho kinase-1
MLCK is a dedicated protein kinase that phosphorylates MLC at Ser-19 and Thr-18 residues (Garcia et al., 1997, Verin et al., 1998a, Verin et al., 1998b). Therefore, we examined its expression in BCEC at protein and mRNA levels. Typical results from Western blotting and RT-PCR are shown in Fig. 1. The two bands in Fig. 1(A) correspond to the smooth muscle (SM-MLCK; MW 130 kDa) and vascular endothelial (EC-MLCK; MW 220 kDa) isoforms of MLCK.
Fig. 1(B) shows expression of MLCK, RhoA, Rho kinase-1,
Discussion
Effects downstream from MLC phosphorylation resulting from acto-myosin interactions and leading to contractility and reorganization of the actin cytoskeleton, appear to be universal (Somlyo and Somlyo, 2000, Turner, 2000, Turner et al., 2000, Kamm and Stull, 2001). Specifically, it is well established that the effects of MLC phosphorylation on the cortical actin cytoskeleton not only alter cell shape but profoundly reduce barrier integrity by opposing the tethering forces that permit
Acknowledgements
Supported by NIH NEI11107 (SPS).
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