Cytoskeletal modulation and tyrosine phosphorylation of tight junction proteins are associated with mainstream cigarette smoke-induced permeability of airway epithelium☆
Introduction
Cigarette smoking causes a vast array of adverse effects, both in the lung itself and distal to this primary site of smoke exposure. Systemic effects include increased incidence of cardiovascular diseases (atherosclerosis, myocardial infarctions, and strokes) and increased susceptibility to infection. Cigarette smoke (CS) exposure is known to increase the permeability of the lung epithelial/endothelial barrier, potentially allowing access of atmospheric components and pathogens to the circulation and leakage of serum and its constituents to the lumen. The mechanisms by which CS disrupts epithelial integrity have not been fully defined but are likely to involve alterations in the function of the tight junctions (TJ), which normally maintain the polarity of the epithelial cells and limit flow of ions and macromolecules from one side of the monolayer to the other. We previously reported that cigarette smoke temporarily increases macromolecular permeability (≈30 min) at focal sites within cultured epithelium by protein tyrosine kinase (PTK)- and Rho kinase (ROCK)-dependent mechanisms. Conversely, the role of myosin light chain kinase (MLCK) was shown to be protective in this system (Olivera et al., 2007). These results suggested that regulation of permeability in this tissue involves actomyosin and protein phosphorylation.
Tyrosine phosphorylation of the TJ proteins has been linked to increased permeability in a number of models. In epidermal carcinoma cells (Van Itallie et al., 1995), phosphorylation of ZO-1 resulted in its redistribution, and in the blood–brain barrier (BBB), tyrosine phosphorylation of occludin caused its redistribution and increased macromolecular permeability (Kago et al., 2006). In an intestinal model, oxidative stress increased tyrosine phosphorylation of occludin and ZO-1, which is associated with loss of ZO-1:occludin complex, loss of TJ:cytoskeleton association, and increased permeability to ions and macromolecules (Rao et al., 2002).
ROCK is an important modulator of the cytoskeleton, known to induce stress fibers (Amano et al., 1997; Fujita et al., 2000), phosphorylate myosin light chain, similar to MLCK (Amano et al., 1996; Kimura et al., 1996); phosphorylate and inactivate the myosin-binding subunit (MBS) of myosin phosphatase (Totsukawa et al., 2000); and phosphorylate Lin-11, Isl-1, and Mec-3 kinase (LIMK) (Ohashi et al., 2000), which inhibits cofilin, the actin-severing factor (Takaishi et al., 2000). The results of these phosphorylation events are the polymerization of filamentous actin at the cell periphery (Takaishi et al., 2000; Bruewer et al., 2004), cytoskeletal contraction through increased net phosphorylation of myosin light chain (Wojciak-Stothard et al., 2001), increased motor activity of myosin heavy chain (Totsukawa et al., 2000), TJ protein redistribution (Stamatovic et al., 2003; Bruewer et al., 2004), and increased permeability (Wojciak-Stothard et al., 2001; Stamatovic et al., 2003).
Our previous data indicated that inhibition of protein tyrosine kinases partially protects barrier function in lung epithelial cells exposed to mainstream smoke (MS) at an air–liquid interface (Olivera et al., 2007). We therefore investigated tyrosine phosphorylation of ZO-1 and occludin and ZO-1:occludin associations. Because inhibition of ROCK also partially protected barrier function, we also examined interactions of the cytoskeleton with the TJ proteins, densities of filamentous actin, and MBS and MLC phosphorylation status following exposure to cigarette smoke.
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Cell culture
Calu-3 adenocarcinoma cells were selected for these experiments due to their airway epithelial origin and ability to form monolayers that are highly impermeable to ionic and macromolecular flux (Wan et al., 2000; Olivera et al., 2007). Cells were obtained from the American Type Cell Collection (Rockville, MD) and used at passages 30–40 for all experiments. Cells to be exposed were seeded 1×105/cm2 into Transwell® inserts, 0.4 μm pore size, 1 cm2 surface area (Costar, Cambridge, MA) and grown in
MS increases filamentous actin
To examine alterations in the localization and polymerization of actin, we exposed cultures to FA or MS, labeled f-actin with rhodamine-phalloidin, and quantified the label by fluorescent microscopic volumetric analysis. MS significantly increased f-actin over controls. Pair-wise comparisons indicated a 52±17% increase in f-actin (p<0.001). Interestingly, it appeared to do so on a cell-to-cell basis, as some cells did not show increased filamentous actin, while others clearly did (Fig. 1A–C).
Actin polymerization correlates with leaks and with redistribution of occludin and ZO-1
Discussion
Air–liquid interface exposure of cultured human lung epithelial cells provides a valuable model system with which to examine mechanisms of effects of aerosols (Aufderheide and Mohr, 2000; Knebel et al., 2002; Seagrave et al., 2007) including cigarette smoke (Aufderheide et al., 2003; Olivera et al., 2007). This model benefits from a direct interaction between the cells and MS in its native form, as would occur at the luminal surface of an intact lung during cigarette smoking. Our previous study
Acknowledgement
The authors gratefully acknowledge the technical assistance of Susan McGrath.
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Cited by (0)
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This work was supported in part by grants from Johnson & Johnson (to C. Knall) and the NIH R03CA91218 (to S.E. Boggs) and by a Grant P30-ES012072 to the University of New Mexico National Institute of Environmental Health Sciences Center.
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Current address: US Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, MD, USA.
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Current address: Sandia National Laboratory, Albuquerque, NM, USA.