Sphingosine-1-Phosphate Promotes Paracellular Junctional Protein Expression and Intestinal Epithelial Cell Barrier Function
Ruiyun Li1,2, Jose Greenspon2, Rao N. Jaladanki1,2, Jian-Ying Wang1,2, Douglas J. Turner*1,2
1Baltimore VAMC, Baltimore, MD; 2University of Maryland, Baltimore, MD
Introduction: Intestinal epithelial barrier dysfunction results from a wide variety of pathologic conditions such as infection, trauma, inflammation and malignancy. Disruption of intercellular junctional proteins appears to be a key pathway to loss of barrier integrity. Preliminary evidence from our lab has shown that Sphingosine-1-phophate (S1P) S1P promotes intestinal epithelial barrier function through a mechanism involving the S1P-1 receptor, leading to increased E-cadherin protein and decreased paracellular permeability. We hypothesized that S1P would increase expression of the tight junction protein occludin in both physiological and pathological conditions. Methods: Studies were performed upon cultured differentiated IECs (IEC-Cdx2L1 line). Western blotting and immunohistochemical staining were performed under standard protocols. Paracellular permeability was assessed using 14C-labelled mannitol, and transepithelial electrical resistance was measured by Voltohmmeter.Results: Intestinal epithelial cells treated with LPS (50 mcM) showed increased paracellular permeability versus control (TEER dropped by ~34%), whereas S1P treatment (0.5 mcM) increased TEER by ~25% versus control. Pretreating IECs with S1P before LPS exposure prevented the LPS-induced drop in TEER in a time-dependent fashion. Treatment of IECs with S1P (0.5 - 5.0 mcM) increased E-cadherin protein by Western blot, however did not similarly increase levels of occludin. However, S1P (0.5- 1.0 mcM) did increase phosphorylated occludin (P-occludin) levels versus control. LPS application (50mcM, 4h) to IECs decreased occludin protein levels by Western blotting that was completely prevented by S1P (0.5 mcM, 1h) pretreatment. Finally, in immunofluourescence studies, LPS (50mcM, 3h) disrupts cortical staining by occludin versus control, however pretreatment with S1P (1mcM, 2h) preserves the occludin cortical staining.Conclusions: These results indicate that S1P promotes intestinal epithelial barrier function during LPS exposure. S1P increases expression of the tight junction protein P-occludin, prevents decreases in occludin levels seen during LPS exposure, and maintains paracellular permeability during LPS exposure.
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