Elsevier

Experimental Eye Research

Volume 79, Issue 4, October 2004, Pages 477-486
Experimental Eye Research

Thrombin-induced phosphorylation of the regulatory light chain of myosin II in cultured bovine corneal endothelial cells

https://doi.org/10.1016/j.exer.2004.06.018Get rights and content

Abstract

Purpose

Phosphorylation of the regulatory light chain of myosin II (referred to as myosin light chain or MLC) leads to a loss of barrier integrity in cellular monolayers by an increase in the contractility of the cortical actin cytoskeleton. This effect has been examined in corneal endothelial (CE) cells.

Methods

Experiments were performed using cultured bovine CE cells (BCEC). MLC phosphorylation was induced by a thrombin-mediated activation of the proteinase-activated receptor-1 (PAR-1). Expression of MLC kinase (MLCK), a Ca2+/calmodulin-dependent protein kinase that phosphorylates MLC at its Ser-19 and Thr-18 residues, was determined by RT-PCR and Western blotting. Expression of PAR-1, RhoA, and Rho kinase-1 (effector of RhoA) was ascertained by RT-PCR. MLC phosphorylation was assessed by urea–glycerol gel electrophoresis followed by immunoblotting. The effects of Rho kinase-1 and PKC were characterized by using their selective inhibitors, Y-27632 and chelerythrine, respectively. Reorganization of the cytoskeleton was evaluated by the phalloidin staining of actin. [Ca2+]i was measured using Fura-2. The barrier integrity was assayed as permeability of BCEC monolayers to horseradish peroxidase (HRP; 44 kDa).

Results

RT-PCR showed expression of MLCK, PAR-1, Rho kinase-1, and RhoA. Western blotting indicated expression of the non-muscle and smooth muscle isoforms of MLCK. Exposure to thrombin induced an increase in [Ca2+]i with the peak unaffected by an absence of extracellular Ca2+. Pre-exposure to thrombin (2 U ml−1; 2 min) led to mono- and di-phosphorylation of MLC. Under both basal conditions and in the presence of thrombin, MLC phosphorylation was prevented by chelerythrine (10 μm) and Y-27632 (<25 μm). Thrombin led to inter-endothelial gaps secondary to the disruption of the cortical actin cytoskeleton, which under resting conditions was organized as a perijunctional actomyosin ring (PAMR). These responses were blocked by pre-treatment with Y-27632. Thrombin also increased permeability to HRP, which was abolished by pre-treatment with Y-27632.

Conclusions

Thrombin induces MLC phosphorylation in BCEC. The consequent increase in the contractility of the actin cytoskeleton produces a centripetal force resulting in inter-endothelial gaps and a breakdown of barrier integrity. These responses are PKC- and Rho kinase-dependent. [Ca2+]i increase, as well as sensitivity of the thrombin response to PKC and Rho kinase inhibitors, are consistent with the expression of PAR-1 receptors in BCEC. Thrombin-induced hyperpermeability is a model to investigate barrier dysfunction induced by MLC phosphorylation.

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 Gαq/11 and Gα12/13 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).

References (41)

  • J.G. Garcia et al.

    Regulation of endothelial cell gap formation and paracellular permeability

    J. Investig. Med.

    (1995)
  • J.G. Garcia et al.

    Regulation of endothelial cell gap formation and barrier dysfunction: role of myosin light chain phosphorylation

    J. Cell. Physiol.

    (1995)
  • J.G. Garcia et al.

    Vascular endothelial cell activation and permeability responses to thrombin

    Blood Coagul. Fibrinolysis

    (1995)
  • J.G. Garcia et al.

    Myosin light chain kinase in endothelium: molecular cloning and regulation

    Am. J. Respir. Cell. Biol.

    (1997)
  • M. Holinstat et al.

    PKCa-induced p115RhoGEF phosphorylation signals endothelial cytoskeletal rearrangement

    J. Biol. Chem.

    (2003)
  • S. Khurana

    Role of actin cytoskeleton in regulation of ion transport: examples from epithelial cells

    J. Membr. Biol.

    (2000)
  • P.V. Rao et al.

    Modulation of aqueous humor outflow facility by the Rho kinase-specific inhibitor Y-27632

    Invest. Ophthalmol. Vis. Sci.

    (2001)
  • M.V. Riley et al.

    Adenosine promotes regulation of corneal hydration through cyclic adenosine monophosphate

    Invest. Ophthalmol. Vis. Sci.

    (1996)
  • M.V. Riley et al.

    Regulation of corneal endothelial barrier function by adenosine, cyclic AMP, and protein kinases

    Invest. Ophthalmol. Vis. Sci.

    (1998)
  • V.P. Sah et al.

    The role of Rho in G protein-coupled receptor signal transduction

    Annu. Rev. Pharmacol. Toxicol.

    (2000)
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