Results
LPA attenuates radiation-induced disruption of tight junctions and
blocks barrier dysfunction in the intestinal epithelium
Caco-2 (Fig. 1, A-D) and m-ICC12 (Fig. 1, E) cell
monolayers were incubated with LPA (10 μM) 30 min before irradiation at
varying doses, and the tight junction integrity was examined by confocal
microscopy for occludin and ZO-1. In control Caco-2 cell monolayers,
occludin, and ZO-1 were co-localized at the intercellular junctions
indicating the presence of intact tight junctions (Fig. 1A). Irradiation
induced a dose-dependent redistribution of tight junction proteins,
whereas pretreatment of the epithelium with LPA attenuated
radiation-induced redistribution of tight junction proteins at both 5 Gy
and 10 Gy doses. Densitometric analysis of ZO-1 fluorescence at the
intercellular junctions indicated a significant reduction in junctional
localization of ZO-1 post-irradiation. LPA-treated samples showed
attenuation of radiation-induced ZO-1 redistribution (Fig. 1B). Tight
junction protein complexes are anchored to the actin cytoskeleton. The
disruption of the actin cytoskeleton is known to disrupt tight junction
(Madara, Stafford, Barenberg & Carlson, 1988). Cofilin is an actin
severing protein that plays an essential role in the disruption of the
actin cytoskeleton. Fluorescence staining for F-actin and
cofilinpS3 (the inactive form) showed that radiation
causes disruption of F-actin filaments and reduces the levels of
cofilinpS3. Pretreatment with LPA blocked
radiation-induced reduction of inactive cofilin and disruption of
F-actin filaments (Fig. 1C). Rho-GTPase is known to be involved in the
activation of cofilin and the promotion of actin cytoskeletal
organization (Vardouli, Moustakas & Stournaras, 2005; Wang, Halasz &
Townes-Anderson, 2019). The data showed that toxin-B, an inhibitor of
Rho-GTPase, blocked LPA-mediated prevention of radiation effects on
cofilin and F-actin organization (Fig. 1D). To confirm the effects of
radiation on the tight junction, we examined the effects of radiation
and LPA in I-mCC12 cell monolayers, a mouse intestinal
epithelial cell line (Bens et al., 1996). Data showed that
radiation-induced a dose-dependent redistribution of ZO-1 from the
intercellular junctions, and LPA pretreatment attenuated this effect of
radiation (Fig. 1E).
Effect of LPA2 deficiency on the radiation-induced
disruption of tight junction and adherens junction in the mouse colon