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Intestinal epithelial barrier dysfunction results from a wide variety of pathologic conditions; at the gastrointestinal mucosal layer cells must be capable of maintaining barrier integrity, and do this through the interplay of multiple active processes. Previous reports from our lab have shown that Sphingosine-1-phophate (S1P) promotes intestinal epithelial barrier function in part through regulation of barrier proteins, and S1P has also been found to be protective in various pathologic states. Lipopolysaccharide (LPS) has been shown to increase paracellular permeability, and recently, to also decrease intracellular S1P. In the current study we hypothesized that S1P would decrease paracellular permeability upon LPS exposure, and would act in part through regulation of caveolin-1 expression
IEC-Cdx2L1 (Cdx) differentiated intestinal epithelial cells were utilized. Western blot analysis, real-time PCR, immunohistochemical staining, were utilized by standard techniques. Transwell permeability to C14-mannitol, FITC-dextran, and measurement of transepithelial electrical resistance (TEER) were utilized for permeability assessments. Sphingosine Kinase 1 (SphK-1) overexpression stable cell lines were selected in rat intestinal epithelial cells (IECs). SphK-1 activity and S1P production were measured by radioactive isotope assay.
LPS-treated (50 mcM) Cdx cells show dramatically increased permeability at 4h, but pretreatment with S1P (0.5 mcM for one hour) was protective of this LPS-induced increase in permeability, and returned permeability to normal levels. S1P also prevented LPS-associated decreases in phosphorylated occludin, and in immunofluorescence studies S1P preserved cortical accumulation of occludin that was disrupted with LPS administration alone. S1P was found to increase levels of toll-like receptor (TLR) 2 in Cdx cells, with no change in levels of TLR4. Similarly, cells stably overexpressing SphK1 demonstrated increased levels of S1P and also increased levels of TLR2 and not TLR4. Cells overexpressing SphK1 and S1P showed dramatically increased plasma membrane levels of Stim1, TRPC1, and the scaffolding protein caveolin-1. Phosphorylated caveolin-1 was significantly decreased with exposure to LPS (5 mcM), however co-treatment with S1P preserved basal caveolin-1 levels. Finally, inhibition of caveolin-1 with siRNA prevented S1P rescue of LPS loss of permeability.
Our findings demonstrate that S1P prevents LPS-associated loss of permeability, and this is in part through its ability to prevent LPS-associated loss of caveolin-1.