Abstract
BackgroundDisruption of epithelial cell-cell adhesions represents an early and important stage in tumor metastasis. This process can be modeled in vitro by exposing cells to chemical tumor promoters, phorbol esters and octylindolactam-V (OI-V), known to activate protein kinase C (PKC). However, molecular events mediating PKC-dependent disruption of epithelial cell-cell contact remain poorly understood. In the present study we investigate mechanisms by which PKC activation induces disassembly of tight junctions (TJs) and adherens junctions (AJs) in a model pancreatic epithelium.ResultsExposure of HPAF-II human pancreatic adenocarcinoma cell monolayers to either OI-V or 12-O-tetradecanoylphorbol-13-acetate caused rapid disruption and internalization of AJs and TJs. Activity of classical PKC isoenzymes was responsible for the loss of cell-cell contacts which was accompanied by cell rounding, phosphorylation and relocalization of the F-actin motor nonmuscle myosin (NM) II. The OI-V-induced disruption of AJs and TJs was prevented by either pharmacological inhibition of NM II with blebbistatin or by siRNA-mediated downregulation of NM IIA. Furthermore, AJ/TJ disassembly was attenuated by inhibition of Rho-associated kinase (ROCK) II, but was insensitive to blockage of MLCK, calmodulin, ERK1/2, caspases and RhoA GTPase.ConclusionOur data suggest that stimulation of PKC disrupts epithelial apical junctions via ROCK-II dependent activation of NM II, which increases contractility of perijunctional actin filaments. This mechanism is likely to be important for cancer cell dissociation and tumor metastasis.
Highlights
Disruption of epithelial cell-cell adhesions represents an early and important stage in tumor metastasis
The aim of this study was to investigate the role of nonmuscle myosin (NM) II in the disassembly of epithelial apical junctions caused by protein kinase C (PKC)-targeting tumor promoters, which mimic the disruption of epithelial cell-cell adhesions during epithelial to mesenchymal transition (EMT) and tumor metastasis
OI-V and TPA rapidly disrupt paracellular barrier and induce disassembly of epithelial apical junctions HPAF-II cells cultured on permeable membrane support rapidly developed confluent cell monolayers with high (1,500–2,100 Ohm × cm2) transepithelial electrical resistance (TEER) (Figure 1)
Summary
Disruption of epithelial cell-cell adhesions represents an early and important stage in tumor metastasis This process can be modeled in vitro by exposing cells to chemical tumor promoters, phorbol esters and octylindolactam-V (OI-V), known to activate protein kinase C (PKC). TJs and AJs mediate cell-cell adhesions through homotypical interactions of their transmembrane proteins such as occludin, claudins and E-cadherin [5,6,7]. These junctional complexes are affiliated with the apical actin cytoskeleton, and participate in outside in transduction of signals and forces [5,8]
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