Journal of the Society for Gynecologic Investigation

 

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Journal of the Society for Gynecologic Investigation, Vol. 13, No. 8, 579-591 (2006)
DOI: 10.1016/j.jsgi.2006.09.002

Non-Muscle Myosin-II-B Filament Regulation of Paracellular Resistance in Cervical Epithelial Cells Is Associated With Modulation of the Cortical Acto-Myosin

Xin Li, MD, PhD

Departments of Reproductive Biology, Physiology and Biophysics, and Oncology, Case Western Reserve University, Cleveland, Ohio

George Gorodeski, MD, PhD

Departments of Reproductive Biology, Physiology and Biophysics, and Oncology, Case Western Reserve University, Cleveland, Ohio; Department of Obstetrics and Gynecology, University Hospitals of Cleveland, 11100 Euclid Ave., Cleveland, OH 44106 gig{at}cwru.edu,george.gorodeski{at}uhhs.com

Objective: To understand myosin regulation of epithelial permeability.

Methods: This was an experimental study, using human cervical epithelial cells CaSki. End points were paracellular permeability (determined in terms of transepithelial electrical resistance); non-muscle myosin-II-B (NMM-II-B) cellular localization; NMM-II-B phosphorylation status; NMM-II-B-actin interaction (determined in vitro by the immunoprecipitation-immunoreactivity method); and NMM-II-B filamentation (determined in vitro using purified NMM-II-B filaments in terms of filaments disassembly/assembly ratios.

Results: Treatment of cells with the Rho-associated kinase (ROCK) inhibitor Y-27632 or with the phosphatase inhibitor okadaic acid decreased the resistance of the lateral intercellular space (RLIS), and increased phosphorylation of NMM-II-B on threonine and serine residues. Y-27632 induced disorganization of the cortical acto-myosin and decreased co-immunoprecipitation of actin with NMM-II-B. Homodimerization assays using NMM-II-B filaments from cells treated with Y-27632 or okadaic acid revealed decreased filamentation compared to control cells. However, okadaic acid blocked Y-27632 decreased filamentation. Treatment with DRB, a casein kinase-II (CK2) inhibitor, induced opposing effects to those of Y-27632 and okadaic acid. Treatment with 5,6-dichloro-1-ß-(D)-ribofuranosylbenzimidazole (DRB) did not involve modulation of actin depolymerization, suggesting that NMM-II-B regulation of the RLIS was independent of actin polymerization status. Exposure of NMM-II-B filaments to CK2 increased filamentation, regardless of prior treatments in vivo with Y-27632, okadaic acid, or DRB.

Conclusions: The results suggest that NMM-II-B filaments are in steady-state equilibrium of phosphorylation-dephosphorylation mediated by CK2 and by ROCK-regulated myosin heavy chain phosphatase, respectively. Increased phosphorylation would tend to inhibit assembly of NMM-II-B filaments and lead to decreased actin-myosin interaction, which would tend to decrease the RLIS and increase the paracellular permeability.

Key Words: Cytoskeleton • dynamic • permeability • actin • human • cervical • vaginal


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