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

Satpathy, Minati; Gallagher, Patricia J.; Lizotte-Waniewski, Michelle; Srinivas, Sangly P.

doi:10.1016/j.exer.2004.06.018

Cited by 61 publications

(80 citation statements)

References 33 publications

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“…1A) as shown previously. 12,13 Treatment with lovastatin alone caused MLC dephosphorylation, and also opposed the thrombin-induced MLC phosphorylation. Densitometric analysis of blots similar to that shown in Figure 1A is summarized in Figure 1B.…”

Section: Immunocytochemistry For F-actin Zo-1 and Diphosphorylated Mlcmentioning

confidence: 99%

“…In our previous studies, 12,13 we demonstrated that thrombin breaks down the barrier integrity of corneal endothelium through RhoA-mediated MLC phophorylation and consequent disruption of the PAMR. In this study, we have investigated whether lovastatin, a lipophilic prototype of statins, can prevent thrombin-induced loss of the barrier integrity in cultured bovine corneal endothelial monolayers.…”

Section: Immunocytochemistry For F-actin Zo-1 and Diphosphorylated Mlcmentioning

confidence: 99%

“…This fi nding is similar to those reported in the vascular endothelium. 3,[11][12][13][14] The actomyosin contraction of the actin cytoskeleton, including that of the PAMR, is increased by phosphorylation of the regulatory light chain of myosin II (also called myosin light chain, or MLC). 15,16 Activation of RhoA, small GTPase of Rho family, is known to increase MLC phosphorylation through its downstream effector, Rho kinase.…”

Section: Immunocytochemistry For F-actin Zo-1 and Diphosphorylated Mlcmentioning

confidence: 99%

“…13 We examined whether lovastatin could mimic the effect of Y-27632. Upon treatment with 10 μM lovastatin for 24 h, the organization of the PAMR is largely unaltered (Fig.…”

Section: Effect Of Lovastatin On the Thrombin-induced Disruption Of Amentioning

confidence: 99%

“…12,13 Cells grown on 60-mm Petri dishes were treated with desired agents, which were followed by an addition of 1 mL of ice-cold lysis buffer containing trichloroacetic acid and dithiothreitol. Protein was extracted using a urea sample buffer containing 8 M urea, 10 mM dithiothreitol, 20 mM Tris base, 23 mM glycine, and 0.04% bromophenol blue.…”

Section: Mlc Phosphorylation Assaymentioning

confidence: 99%

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Lovastatin Inhibits the Thrombin-Induced Loss of Barrier Integrity in Bovine Corneal Endothelium

Shivanna

Jalimarada

Srinivas

2010

Journal of Ocular Pharmacology and Therapeutics

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Purpose: Increased actomyosin contraction of the dense band of actin cytoskeleton at the apical junctional complex (perijunctional actomyosin ring, PAMR) breaks down the barrier integrity of corneal endothelium. This study has investigated the effi cacy of statins, which inhibit activation of RhoA, in opposing the thrombininduced loss of barrier integrity of monolayers of cultured bovine corneal endothelium. Methods: Myosin light chain (MLC) phosphorylation, a biochemical measure of actomyosin contraction, was assayed by urea-glycerol gel electrophoresis, followed by western blot analysis. The locus of MLC phosphorylation and changes in the organization of the PAMR were visualized by immunostaining. Phosphorylation of MYPT1, a regulatory subunit of myosin light-chain phosphatase (MLCP), was assessed by Western blot analysis to determine down-regulation of RhoA. The barrier integrity was assessed in terms of trans-endothelial electrical resistance (TER), and further confi rmed by determining permeability to FITC dextran (10 kDa) and distribution of ZO-1, a marker of tight junctional assembly. Results: Lovastatin, a prototype of lipophilic statins, induced MLC dephosphorylation under basal conditions. It opposed increase in phosphorylation of MLC and MYPT1 in response to thrombin and nocodazole, agents known to activate RhoA in the endothelium. Pretreatment with the statin opposed the thrombin-and nocodazole-induced disruption of the PAMR and the thrombin-induced decline in TER. Lovastatin also opposed the thrombin-and nocodazole-induced increase in permeability to FITC dextran and redistribution of ZO-1. However, upon supplementation with GGPP (geranylgeranyl pyrophosphate), lovastatin failed to oppose the effects of thrombin and nocodazole on the PAMR, ppMLC, and ZO-1 distribution. Conclusions: Lovastatin attenuates RhoA activation in the corneal endothelium presumably by reducing its isoprenylation. This underlies the suppression of the thrombin-induced loss in barrier integrity of the corneal endothelium.

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Section: Immunocytochemistry For F-actin Zo-1 and Diphosphorylated Mlcmentioning

confidence: 99%

Section: Immunocytochemistry For F-actin Zo-1 and Diphosphorylated Mlcmentioning

confidence: 99%

Section: Immunocytochemistry For F-actin Zo-1 and Diphosphorylated Mlcmentioning

confidence: 99%

“…13 We examined whether lovastatin could mimic the effect of Y-27632. Upon treatment with 10 μM lovastatin for 24 h, the organization of the PAMR is largely unaltered (Fig.…”

Section: Effect Of Lovastatin On the Thrombin-induced Disruption Of Amentioning

confidence: 99%

Section: Mlc Phosphorylation Assaymentioning

confidence: 99%

See 3 more Smart Citations

Lovastatin Inhibits the Thrombin-Induced Loss of Barrier Integrity in Bovine Corneal Endothelium

Shivanna

Jalimarada

Srinivas

2010

Journal of Ocular Pharmacology and Therapeutics

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Thrombin promotes actin stress fiber formation in RPE through Rho/ROCK‐mediated MLC phosphorylation

Ruiz-Loredo

López

López-Colomé

2010

Journal Cellular Physiology

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The retinal pigment epithelium (RPE) forms the outer blood-retina barrier (BRB). Most retinal diseases involve BRB breakdown, whereupon thrombin contained in serum directly contacts the RPE. Thrombin is known to promote actin stress fiber formation, an important determinant in eye diseases involving the epithelial-mesenchymal transition (EMT) and migration of RPE cells, such as proliferative vitreoretinopathy. We analyzed thrombin effect on signaling pathways leading to myosin light chain (MLC) phosphorylation and actin stress fiber formation in primary cultures of rat RPE cells, in order to support a role for thrombin in RPE transdifferentiation. MLC phosphorylation was measured by Western blot; actin cytoskeleton was visualized using immunofluorescent phalloidin, and Rho GTPase activation was assessed by ELISA. We showed that thrombin/PAR-1 induces the time- and dose-dependent phosphorylation of MLC through the activation of Rho/ROCK and myosin light chain kinase (MLCK). ROCK increased phospho-MLC by phosphorylating MLC and by inhibiting MLC phosphatase. Thrombin effect was abolished by the ROCK inhibitor Y-27632, whereas MLCK inhibitor ML-7 and PLC-β inhibitor U73122 attenuated MLC phosphorylation by ≈50%, suggesting the activation of MLCK by PLC-β-mediated calcium increase. Additionally, thrombin-induced MLC phosphorylation was blocked by the inhibitory PKCζ pseudosubstrate, wortmannin, and LY294002, indicating IP(3)/PKCζ involvement in the control of MLC phosphorylation. Moreover, we demonstrated that thrombin effect on MLC induces actin stress fiber formation, since this effect was prevented by inhibiting the pathways leading to MLC phosphorylation. We conclude that thrombin stimulation of MLC phosphorylation and actin stress fiber formation may be involved in thrombin-induced RPE cell transformation subsequent to BRB dysfunction.

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Signaling pathways in cytoskeletal responses to plasma membrane depolarization in corneal endothelial cells

Evans

Hernández

Chifflet

2019

Journal Cellular Physiology

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In previous work, we reported that plasma membrane potential depolarization (PMPD) provokes cortical F-actin remodeling in bovine corneal endothelial (BCE) cells in culture, which eventually leads to the appearance of intercellular gaps. In kidney epithelial cells it has been shown that PMPD determines an extracellularsignal-regulated kinase (ERK)/Rho-dependent increase in diphosphorylated myosin light chain (ppMLC). The present study investigated the signaling pathways involved in the response of BCE cells to PMPD. Differently to renal epithelial cells, we observed that PMPD leads to a decrease in monophosphorylated MLC (pMLC) without affecting diphosphorylated MLC. Also, that the pMLC reduction is a consequence of cyclic adenosine 3′,5′-monophosphate (cAMP)/protein kinase A (PKA) activation. In addition, we found evidence that the cAMP increase mostly depends on soluble adenylyl cyclase activity. Inhibition of this enzyme reduces the effect of PMPD on the cAMP rise, F-actin remodeling, and pMLC decrease. No changes in phosho-ERK were observed, although we could determine that RhoA undergoes activation. Our results suggested that active RhoA is not involved in the intercellular gap formation. Overall, the findings of this study support the view that, differently to renal epithelial cells, in BCE cells PMPD determines cytoskeletal reorganization via activation of the cAMP/PKA pathway. K E Y W O R D S actin cytoskeleton, cAMP/PKA, corneal endothelium, myosin light chain, plasma membrane depolarization

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

Cited by 61 publications

References 33 publications

Lovastatin Inhibits the Thrombin-Induced Loss of Barrier Integrity in Bovine Corneal Endothelium

Lovastatin Inhibits the Thrombin-Induced Loss of Barrier Integrity in Bovine Corneal Endothelium

Thrombin promotes actin stress fiber formation in RPE through Rho/ROCK‐mediated MLC phosphorylation

Signaling pathways in cytoskeletal responses to plasma membrane depolarization in corneal endothelial cells

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