Digestive Diseases and Sciences

, Volume 64, Issue 2, pp 421–431 | Cite as

Osteopontin Protects Colonic Mucosa from Dextran Sodium Sulfate-Induced Acute Colitis in Mice by Regulating Junctional Distribution of Occludin

  • Sang-Ho Woo
  • Su-Hyung Lee
  • Jun-Won Park
  • Du-Min Go
  • Dae-Yong KimEmail author
Original Article



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.


To investigate the role of OPN on tight junctions.


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.


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.


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.


Mucosal protection Osteopontin Occludin Tight junction 



This study was supported by the Research Institute of Veterinary Science, College of Veterinary Medicine, Seoul National University and by the Korea Mouse Phenotyping Project (NRF-2016M3A9D5A01952416) of a National Research Foundation grant funded by the Korean government (MSIP).

Compliance with ethical standards

Conflict of interest

All authors declare that they have no conflict of interest.

Supplementary material

10620_2018_5246_MOESM1_ESM.docx (84 kb)
Supplementary material 1 (DOCX 83 kb)


  1. 1.
    Balda MS, Matter K. Tight junctions at a glance. J Cell Sci. 2008;121:3677–3682.CrossRefGoogle Scholar
  2. 2.
    Lee SH. Intestinal permeability regulation by tight junction: implication on inflammatory bowel diseases. Interest Res. 2015;13:11–18.CrossRefGoogle Scholar
  3. 3.
    Rao R. Occludin phosphorylation in regulation of epithelial tight junctions. Ann N Y Acad Sci. 2009;1165:62–68.CrossRefGoogle Scholar
  4. 4.
    Saitou M, Fujimoto K, Doi Y, et al. Occludin-deficient embryonic stem cells can differentiate into polarized epithelial cells bearing tight junctions. J Cell Biol. 1998;141:397–408.CrossRefGoogle Scholar
  5. 5.
    Wong V, Gumbiner BM. A synthetic peptide corresponding to the extracellular domain of occludin perturbs the tight junction permeability barrier. J Cell Biol. 1997;136:399–409.CrossRefGoogle Scholar
  6. 6.
    Mir H, Meena AS, Chaudhry KK, et al. Occludin deficiency promotes ethanol-induced disruption of colonic epithelial junctions, gut barrier dysfunction and liver damage in mice. Biochim Biophys Acta. 2016;1860:765–774.CrossRefGoogle Scholar
  7. 7.
    Sase SP, Nagane N, Ganu JV. Osteopontin: a novel protein molecule. Ind Med Gaz. 2012;146:62–66.Google Scholar
  8. 8.
    Sodek J, Batista Da Silva AP, Zohar R. Osteopontin and mucosal protection. J Dent Res. 2006;85:404–415.CrossRefGoogle Scholar
  9. 9.
    Tang R, Yang G, Zhang S, Wu C, Chen M. Opposite effects of interferon regulatory factor 1 and osteopontin on the apoptosis of epithelial cells induced by TNF-α in inflammatory bowel disease. Inflamm Bowel Dis. 2014;20:1950–1961.CrossRefGoogle Scholar
  10. 10.
    Da Silva APB, Ellen RP, Sørensen ES, Goldberg HA, Zohar R, Sodek J. Osteopontin attenuation of dextran sulfate sodium-induced colitis in mice. Lab Investig. 2009;89:1169–1181.CrossRefGoogle Scholar
  11. 11.
    Da Silva APB, Pollett A, Rittling SR, Denhardt DT, Sodek J, Zohar R. Exacerbated tissue destruction in DSS-induced acute colitis of OPN-null mice is associated with downregulation of TNF-α expression and non-programmed cell death. J Cell Phys. 2006;208:629–639.CrossRefGoogle Scholar
  12. 12.
    Heilmann K, Hoffmann U, Witte E, et al. Osteopontin as two-sided mediator of intestinal inflammation. J Cell Mol Med. 2009;13:1162–1174.CrossRefGoogle Scholar
  13. 13.
    Suzuki H, Ayer R, Sugawara T, et al. Protective effects of recombinant osteopontin on early brain injury after subarachnoid hemorrhage in rats. Crit Care Med. 2010;38:612–618.CrossRefGoogle Scholar
  14. 14.
    Ge X, Lu Y, Leung TM, Sørensen ES, Nieto N. Milk osteopontin, a nutritional approach to prevent alcohol-induced liver injury. Am J Physiol Gastrointest Liver Physiol. 2013;304:G929–G939.CrossRefGoogle Scholar
  15. 15.
    Gommeaux J, Cano C, Garcia S, et al. Colitis and colitis-associated cancer are exacerbated in mice deficient for tumor protein 53-induced nuclear protein 1. Mol Cell Biol. 2007;27:2215–2228.CrossRefGoogle Scholar
  16. 16.
    Okayasu I, Hatakeyama S, Yamada M, Ohkusa T, Inagaki Y, Nakaya R. A novel method in the induction of reliable experimental acute and chronic ulcerative colitis in mice. Gastroenterology. 1990;98:694–702.CrossRefGoogle Scholar
  17. 17.
    Moolenbeek C, Ruitenberg E. The ‘Swiss roll’: a simple technique for histological studies of the rodent intestine. Lab Anim. 1981;15:57–60.CrossRefGoogle Scholar
  18. 18.
    Cooper HS, Murthy S, Shah R, Sedergran D. Clinicopathologic study of dextran sulfate sodium experimental murine colitis. Lab Investig. 1993;69:238–249.Google Scholar
  19. 19.
    Elamin E, Masclee A, Dekker J, Jonkers D. Ethanol disrupts intestinal epithelial tight junction integrity through intracellular calcium-mediated Rho/ROCK activation. Am J Physiol Gastrointest Liver Physiol. 2014;306:G677–G685.CrossRefGoogle Scholar
  20. 20.
    Wong V. Phosphorylation of occludin correlates with occludin localization and function at the tight junction. Am J Physiol. 1997;273:C1859–C1867.CrossRefGoogle Scholar
  21. 21.
    Hidalgo IJ, Raub TJ, Borchardt RT. Characterization of the human colon carcinoma cell line (Caco-2) as a model system for intestinal epithelial permeability. Gastroenterology. 1989;96:736–749.CrossRefGoogle Scholar
  22. 22.
    Lee SD, Osei-Twum JA, Wasan KM. Dose-dependent targeted suppression of P-glycoprotein expression and function in Caco-2 cells. Mol Pharm. 2013;10:2323–2330.CrossRefGoogle Scholar
  23. 23.
    Pinto M. Enterocyte-like differentiation and polarization of the human colon carcinoma cell line Caco-2 in culture. Biol Cell. 1983;47:323–330.Google Scholar
  24. 24.
    Chen F, Liu H, Shen Q, et al. Osteopontin: participation in inflammation or mucosal protection in inflammatory bowel diseases? Dig Dis Sci. 2013;58:1569–1580. Scholar
  25. 25.
    Gassler N, Autschbach F, Gauer S, et al. Expression of osteopontin (Eta-1) in Crohn disease of the terminal ileum. Scand J Gastroenterol. 2002;37:1286–1295.CrossRefGoogle Scholar
  26. 26.
    Ashkar S, Weber GF, Panoutsakopoulou V, et al. Eta-1 (osteopontin): an early component of type-1 (cell-mediated) immunity. Science. 2000;287:860–864.CrossRefGoogle Scholar
  27. 27.
    Park JW, Lee SH, Kim HK, Kwon HJ, Kim DY. Osteopontin depletion decreases inflammation and gastric epithelial proliferation during Helicobacter pylori infection in mice. Lab Investig. 2015;95:660–671.CrossRefGoogle Scholar
  28. 28.
    Sato T, Nakai T, Tamura N, et al. Osteopontin/Eta-1 upregulated in Crohn’s disease regulates the Th1 immune response. Gut. 2005;54:1254–1262.CrossRefGoogle Scholar
  29. 29.
    Zhong J, Eckhardt ER, Oz HS, Bruemmer D, de Villiers WJ. Osteopontin deficiency protects mice from Dextran sodium sulfate-induced colitis. Inflamm Bowel Dis. 2006;12:790–796.CrossRefGoogle Scholar
  30. 30.
    D’Incà R, De Leo V, Corrao G, et al. Intestinal permeability test as a predictor of clinical course in Crohn’s disease. Am J Gastroenterol. 1999;94:2956.CrossRefGoogle Scholar
  31. 31.
    Kitajima S, Takuma S, Morimoto M. Changes in colonic mucosal permeability in mouse colitis induced with dextran sulfate sodium. Exp Anim. 1999;48:137–143.CrossRefGoogle Scholar
  32. 32.
    Ni J, Chen S, Hollander D. Effects of dextran sulphate sodium on intestinal epithelial cells and intestinal lymphocytes. Gut. 1996;39:234–241.CrossRefGoogle Scholar
  33. 33.
    Poritz LS, Garver KI, Green C, Fitzpatrick L, Ruggiero F, Koltun WA. Loss of the tight junction protein ZO-1 in dextran sulfate sodium induced colitis. J Surg Res. 2007;140:12–19.CrossRefGoogle Scholar
  34. 34.
    Sakakibara A, Furuse M, Saitou M, Ando-Akatsuka Y, Tsukita S. Possible involvement of phosphorylation of occludin in tight junction formation. J Cell Biol. 1997;137:1393–1401.CrossRefGoogle Scholar
  35. 35.
    Rao RK, Basuroy S, Rao VU, Karnaky KJ Jr, Gupta A. Tyrosine phosphorylation and dissociation of occludin-ZO-1 and E-cadherin-beta-catenin complexes from the cytoskeleton by oxidative stress. Biochem J. 2002;368:471–481.CrossRefGoogle Scholar
  36. 36.
    Banan A, Zhang L, Farhadi A, et al. Critical role of the atypical λ isoform of protein kinase C (PKC-λ) in oxidant-induced disruption of the microtubule cytoskeleton and barrier function of intestinal epithelium. J Pharmacol Exp Ther. 2005;312:458–471.CrossRefGoogle Scholar
  37. 37.
    Clarke H, Soler AP, Mullin JM. Protein kinase C activation leads to dephosphorylation of occludin and tight junction permeability increase in LLC-PK1 epithelial cell sheets. J Cell Sci. 2000;113:3187–3196.Google Scholar
  38. 38.
    Jain S, Suzuki T, Seth A, Samak G, Rao R. Protein kinase Cζ phosphorylates occludin and promotes assembly of epithelial tight junctions. Biochem J. 2011;437:289–299.CrossRefGoogle Scholar
  39. 39.
    Nunbhakdi-Craig V, Machleidt T, Ogris E, Bellotto D, White CL, Sontag E. Protein phosphatase 2A associates with and regulates atypical PKC and the epithelial tight junction complex. J Cell Biol. 2002;158:967–978.CrossRefGoogle Scholar
  40. 40.
    Seth A, Sheth P, Elias BC, Rao R. Protein phosphatases 2A and 1 interact with occludin and negatively regulate the assembly of tight junctions in the CACO-2 cell monolayer. J Biol Chem. 2007;282:11487–11498.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Sang-Ho Woo
    • 1
  • Su-Hyung Lee
    • 2
  • Jun-Won Park
    • 3
  • Du-Min Go
    • 1
  • Dae-Yong Kim
    • 1
    Email author
  1. 1.Department of Veterinary Pathology, College of Veterinary MedicineSeoul National UniversitySeoulSouth Korea
  2. 2.Branch of Carcinogenesis and MetastasisResearch Institute of National Cancer CenterGoyangSouth Korea
  3. 3.Division of GeneticsCancer Research Institute, Kanazawa UniversityKanazawaJapan

Personalised recommendations