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Thrombin stimulates stress fiber assembly in RPE cells by PKC/CPI-17-mediated MLCP inactivation.
Exp Eye Res. 2012 Mar; 96(1):13-23.EE

Abstract

Most retinal proliferative diseases involve blood-retinal barrier (BRB) breakdown, exposing the retinal pigment epithelium (RPE) to thrombin, which triggers cell transformation, proliferation and migration through the activation of PAR-1. These processes require the assembly of contractile stress fibers containing actin and non-muscle myosin II, which allow cell movement upon phosphorylation of the myosin light chains (MLCs). PKC family of kinases promotes agonist-mediated contraction in smooth muscle and endothelial cells through the activation of its downstream target, the PKC-potentiated inhibitory protein of 17 kDa (CPI-17), which specifically inhibits MLC phosphatase. Although the participation of PKC in RPE cell transdifferentiation has been suggested, the role of PKC/CPI-17 signaling has not been investigated. The purpose of this study was to analyze the involvement of specific PKC isoenzymes and their effector protein CPI-17 in thrombin-induced MLC phosphorylation and actin stress fiber assembly in RPE cells. Rat RPE cells in primary culture were shown to respond to thrombin stimulation by activation of conventional, novel and atypical PKC isoforms and the downstream phosphorylation of CPI-17 and MLC, which in turn promoted actin stress fiber assembly. These effects were prevented by the pharmacological inhibition of conventional PKC isoenzymes (Ro-32-0432) and novel PKCδ (rottlerin and δV1-1 antagonist peptide), as well as by myristoylated pseudosubstrates specifically directed to conventional and atypical PKC isoforms. Thrombin effects were mimicked by phorbol 12-myristate 13-acetate (PMA), further confirming the involvement of diacylglycerol (DAG)-sensitive classical and novel PKC isoforms in thrombin-induced actin cytoskeleton modification. The present work shows, for the first time, the functional expression of the oncoprotein CPI-17 in RPE cells and suggests that PKC/CPI-17 signaling is involved in the control of actin cytoskeletal remodeling leading to cell motility in RPE cells exposed to thrombin, and hence could contribute to the development of proliferative eye diseases.

Authors+Show Affiliations

División de Neurociencias, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, D.F., México, Mexico.No affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

22300615

Citation

Ruiz-Loredo, Ariadna Yolanda, et al. "Thrombin Stimulates Stress Fiber Assembly in RPE Cells By PKC/CPI-17-mediated MLCP Inactivation." Experimental Eye Research, vol. 96, no. 1, 2012, pp. 13-23.
Ruiz-Loredo AY, López E, López-Colomé AM. Thrombin stimulates stress fiber assembly in RPE cells by PKC/CPI-17-mediated MLCP inactivation. Exp Eye Res. 2012;96(1):13-23.
Ruiz-Loredo, A. Y., López, E., & López-Colomé, A. M. (2012). Thrombin stimulates stress fiber assembly in RPE cells by PKC/CPI-17-mediated MLCP inactivation. Experimental Eye Research, 96(1), 13-23. https://doi.org/10.1016/j.exer.2012.01.008
Ruiz-Loredo AY, López E, López-Colomé AM. Thrombin Stimulates Stress Fiber Assembly in RPE Cells By PKC/CPI-17-mediated MLCP Inactivation. Exp Eye Res. 2012;96(1):13-23. PubMed PMID: 22300615.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Thrombin stimulates stress fiber assembly in RPE cells by PKC/CPI-17-mediated MLCP inactivation. AU - Ruiz-Loredo,Ariadna Yolanda, AU - López,Edith, AU - López-Colomé,Ana María, Y1 - 2012/01/25/ PY - 2011/10/21/received PY - 2012/01/11/revised PY - 2012/01/17/accepted PY - 2012/2/4/entrez PY - 2012/2/4/pubmed PY - 2012/5/12/medline SP - 13 EP - 23 JF - Experimental eye research JO - Exp. Eye Res. VL - 96 IS - 1 N2 - Most retinal proliferative diseases involve blood-retinal barrier (BRB) breakdown, exposing the retinal pigment epithelium (RPE) to thrombin, which triggers cell transformation, proliferation and migration through the activation of PAR-1. These processes require the assembly of contractile stress fibers containing actin and non-muscle myosin II, which allow cell movement upon phosphorylation of the myosin light chains (MLCs). PKC family of kinases promotes agonist-mediated contraction in smooth muscle and endothelial cells through the activation of its downstream target, the PKC-potentiated inhibitory protein of 17 kDa (CPI-17), which specifically inhibits MLC phosphatase. Although the participation of PKC in RPE cell transdifferentiation has been suggested, the role of PKC/CPI-17 signaling has not been investigated. The purpose of this study was to analyze the involvement of specific PKC isoenzymes and their effector protein CPI-17 in thrombin-induced MLC phosphorylation and actin stress fiber assembly in RPE cells. Rat RPE cells in primary culture were shown to respond to thrombin stimulation by activation of conventional, novel and atypical PKC isoforms and the downstream phosphorylation of CPI-17 and MLC, which in turn promoted actin stress fiber assembly. These effects were prevented by the pharmacological inhibition of conventional PKC isoenzymes (Ro-32-0432) and novel PKCδ (rottlerin and δV1-1 antagonist peptide), as well as by myristoylated pseudosubstrates specifically directed to conventional and atypical PKC isoforms. Thrombin effects were mimicked by phorbol 12-myristate 13-acetate (PMA), further confirming the involvement of diacylglycerol (DAG)-sensitive classical and novel PKC isoforms in thrombin-induced actin cytoskeleton modification. The present work shows, for the first time, the functional expression of the oncoprotein CPI-17 in RPE cells and suggests that PKC/CPI-17 signaling is involved in the control of actin cytoskeletal remodeling leading to cell motility in RPE cells exposed to thrombin, and hence could contribute to the development of proliferative eye diseases. SN - 1096-0007 UR - https://www.unboundmedicine.com/medline/citation/22300615/Thrombin_stimulates_stress_fiber_assembly_in_RPE_cells_by_PKC/CPI_17_mediated_MLCP_inactivation_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0014-4835(12)00033-4 DB - PRIME DP - Unbound Medicine ER -