Sang-Ho Woo1, Su-Hyung Lee2, Jun-Won Park3, Du-Min Go1, Dae-Yong Kim4. 1. Department of Veterinary Pathology, College of Veterinary Medicine, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul, 08826, South Korea. 2. Branch of Carcinogenesis and Metastasis, Research Institute of National Cancer Center, Goyang, Gyeonggi, 10408, South Korea. 3. Division of Genetics, Cancer Research Institute, Kanazawa University, Kanazawa, 920-1192, Japan. 4. Department of Veterinary Pathology, College of Veterinary Medicine, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul, 08826, South Korea. daeyong@snu.ac.kr.
Abstract
BACKGROUND: Osteopontin (OPN) has been reported to play an important role in intestinal mucosal protection. Although OPN may have positive effects on tight junctions, the exact relationship between OPN and tight junctions has yet to be elucidated. AIMS: To investigate the role of OPN on tight junctions. METHODS: We evaluated clinical signs and histopathology of acute colitis induced by dextran sodium sulfate (DSS) in OPN knockout and wild-type (WT) mice in vivo. Expression levels of occludin and zonula occludens-1 were examined using immunofluorescence. For in vitro analysis, an siRNA-mediated OPN-suppressed Caco-2 monolayer was used. Expression levels and patterns of occludin were analyzed by immunofluorescence, and transepithelial electrical resistance (TER) was measured to evaluate barrier function. Triton X-100 fractionation was used to analyze phosphorylated occludin associated with tight junctional localization. RESULTS: OPN deficiency resulted in an elevated disease activity index, shortened colon length, and aggravated histological signs in mice with DSS-induced acute colitis compared to WT mice. OPN deficiency decreased occludin expression in the colonic mucosa. In Caco-2 monolayers, OPN suppression reduced junctional occludin and redistributed it into the intracellular compartment with decreased TER. Furthermore, western blot for occludin from Triton X-100 insoluble fraction revealed that OPN suppression reduced the phosphorylated form of occludin, which is actually distributed in the tight junction. CONCLUSIONS: Our study showed that OPN is essential for maintaining the tight junction complex by allowing occludin to localize at tight junctions. This could constitute additional evidence that OPN plays a crucial role in intestinal mucosal protection.
BACKGROUND:Osteopontin (OPN) has been reported to play an important role in intestinal mucosal protection. Although OPN may have positive effects on tight junctions, the exact relationship between OPN and tight junctions has yet to be elucidated. AIMS: To investigate the role of OPN on tight junctions. METHODS: We evaluated clinical signs and histopathology of acute colitis induced by dextran sodium sulfate (DSS) in OPN knockout and wild-type (WT) mice in vivo. Expression levels of occludin and zonula occludens-1 were examined using immunofluorescence. For in vitro analysis, an siRNA-mediated OPN-suppressed Caco-2 monolayer was used. Expression levels and patterns of occludin were analyzed by immunofluorescence, and transepithelial electrical resistance (TER) was measured to evaluate barrier function. Triton X-100 fractionation was used to analyze phosphorylated occludin associated with tight junctional localization. RESULTS:OPN deficiency resulted in an elevated disease activity index, shortened colon length, and aggravated histological signs in mice with DSS-induced acute colitis compared to WT mice. OPN deficiency decreasedoccludin expression in the colonic mucosa. In Caco-2 monolayers, OPN suppression reduced junctional occludin and redistributed it into the intracellular compartment with decreased TER. Furthermore, western blot for occludin from Triton X-100 insoluble fraction revealed that OPN suppression reduced the phosphorylated form of occludin, which is actually distributed in the tight junction. CONCLUSIONS: Our study showed that OPN is essential for maintaining the tight junction complex by allowing occludin to localize at tight junctions. This could constitute additional evidence that OPN plays a crucial role in intestinal mucosal protection.
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