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