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Par-3 modulates intestinal epithelial barrier function through regulating intracellular trafficking of occludin and myosin light chain phosphorylation

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Abstract

Background

Tight junctions play a critical role in the maintenance of intestinal barrier function. Partitioning-defective protein 3 (Par-3) can regulate intestinal barrier function through the modulation of tight junction assembly and cell polarity. However, the mechanisms are still not fully understood.

Methods

Adult C57BL/6 mice were treated with dextran sulfate sodium for 7 days, and segments of colon were harvested for immunofluorescent staining of Par-3. Caco-2 intestinal epithelial cells were treated with tumor necrosis factor α (TNF-α) for 24 h, and Par-3 expression was detected by Western blot analysis and immunofluorescence. Additionally, Caco-2 cells were treated with Par-3 small interfering RNA, and altered expression and subcellular localization of tight junction proteins were studied by Western blot analysis and immunofluorescence. Furthermore, the interaction between Par-3 and myosin light chain (MLC) was detected by immunoprecipitation.

Results

Par-3 was downregulated in murine dextran sulfate sodium induced acute inflammation and TNF-α-treated Caco-2 cells. Depletion of Par-3 expression by small interfering RNA delayed intestinal epithelial barrier development in Caco-2 cells. This regulation was due to the redistribution of the tight junction protein occludin rather than the altered total levels of tight junction proteins. Par-3 silencing blocked the trafficking of occludin from or through the Golgi complex to the cell surface, and dramatically induced occludin accumulated at the Golgi complex. Importantly, Par-3 can interact with MLC, and loss of Par-3 upregulated MLC kinase expression and MLC phosphorylation, which contributed to intestinal epithelial barrier dysfunction.

Conclusions

These results indicate that Par-3 plays an important role in the modulation of intestinal barrier function by regulating delivery of occludin as well as suppression of MLC phosphorylation.

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References

  1. Cunningham KE, Turner JR. Myosin light chain kinase: pulling the strings of epithelial tight junction function. Ann N Y Acad Sci. 2012;1258:34–42.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  2. Nusrat A, Turner JR, Madara JL. Molecular physiology and pathophysiology of tight junctions. IV. Regulation of tight junctions by extracellular stimuli: nutrients, cytokines, and immune cells. Am J Physiol Gastrointest Liver Physiol. 2000;279:G851–7.

    CAS  PubMed  Google Scholar 

  3. Turner JR. Molecular basis of epithelial barrier regulation: from basic mechanisms to clinical application. Am J Pathol. 2006;169:1901–9.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  4. Visser J, Rozing J, Sapone A, et al. Tight junctions, intestinal permeability, and autoimmunity: celiac disease and type 1 diabetes paradigms. Ann N Y Acad Sci. 2009;1165:195–205.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  5. Gonzalez-Mariscal L, Betanzos A, Nava P, et al. Tight junction proteins. Prog Biophys Mol Biol. 2003;81:1–44.

    Article  CAS  PubMed  Google Scholar 

  6. Paris L, Tonutti L, Vannini C, et al. Structural organization of the tight junctions. Biochim Biophys Acta. 2008;1778:646–59.

    Article  CAS  PubMed  Google Scholar 

  7. Shin K, Fogg VC, Margolis B. Tight junctions and cell polarity. Annu Rev Cell Dev Biol. 2006;22:207–35.

    Article  CAS  PubMed  Google Scholar 

  8. Mankertz J, Schulzke JD. Altered permeability in inflammatory bowel disease: pathophysiology and clinical implications. Curr Opin Gastroenterol. 2007;23:379–83.

    Article  CAS  PubMed  Google Scholar 

  9. Stallmach A, Giese T, Schmidt C, et al. Cytokine/chemokine transcript profiles reflect mucosal inflammation in Crohn’s disease. Int J Colorectal Dis. 2004;19:308–15.

    Article  PubMed  Google Scholar 

  10. Andoh A, Yagi Y, Shioya M, et al. Mucosal cytokine network in inflammatory bowel disease. World J Gastroenterol. 2008;14:5154–61.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  11. Bruewer M, Luegering A, Kucharzik T, et al. Proinflammatory cytokines disrupt epithelial barrier function by apoptosis-independent mechanisms. J Immunol. 2003;171:6164–72.

    Article  CAS  PubMed  Google Scholar 

  12. Koch S, Nusrat A. Dynamic regulation of epithelial cell fate and barrier function by intercellular junctions. Ann N Y Acad Sci. 2009;1165:220–7.

    Article  CAS  PubMed  Google Scholar 

  13. Marano CW, Lewis SA, Garulacan LA, et al. Tumor necrosis factor-alpha increases sodium and chloride conductance across the tight junction of CACO-2 BBE, a human intestinal epithelial cell line. J Membr Biol. 1998;161:263–74.

    Article  CAS  PubMed  Google Scholar 

  14. Youakim A, Ahdieh M. Interferon-γ decreases barrier function in T84 cells by reducing ZO-1 levels and disrupting apical actin. Am J Physiol. 1999;276:G1279–88.

    CAS  PubMed  Google Scholar 

  15. Bruewer M, Utech M, Ivanov AI, et al. Interferon-gamma induces internalization of epithelial tight junction proteins via a macropinocytosis-like process. FASEB J. 2005;19:923–33.

    Article  CAS  PubMed  Google Scholar 

  16. Yang S, Yu M, Sun L, et al. Interferon-γ-induced intestinal epithelial barrier dysfunction by NF-κB/HIF-1α pathway. J Interferon Cytokine Res. 2014;34:195–203.

    Article  CAS  PubMed  Google Scholar 

  17. Schumann M, Gunzel D, Buergel N, et al. Cell polarity-determining proteins Par-3 and PP-1 are involved in epithelial tight junction defects in coeliac disease. Gut. 2012;61:220–8.

    Article  CAS  PubMed  Google Scholar 

  18. Joberty G, Petersen C, Gao L, et al. The cell-polarity protein Par-6 links Par-3 and atypical protein kinase C to Cdc42. Nat Cell Biol. 2000;2:531–9.

    Article  CAS  PubMed  Google Scholar 

  19. Wapenaar MC, Monsuur AJ, van Bodegraven AA, et al. Associations with tight junction genes PARD3 and MAGI2 in Dutch patients point to a common barrier defect for coeliac disease and ulcerative colitis. Gut. 2008;57:463–7.

    Article  CAS  PubMed  Google Scholar 

  20. Chen X, Macara IG. Par-3 controls tight junction assembly through the Rac exchange factor Tiam1. Nat Cell Biol. 2005;7:262–9.

    Article  CAS  PubMed  Google Scholar 

  21. Wells CL, van de Westerlo EM, Jechorek RP, et al. Cytochalasin-induced actin disruption of polarized enterocytes can augment internalization of bacteria. Infect Immun. 1998;66:2410–9.

    PubMed Central  CAS  PubMed  Google Scholar 

  22. Melgar S, Karlsson L, Rehnstrom E, et al. Validation of murine dextran sulfate sodium-induced colitis using four therapeutic agents for human inflammatory bowel disease. Int Immunopharmacol. 2008;8:836–44.

    Article  CAS  PubMed  Google Scholar 

  23. Fries W, Muja C, Crisafulli C, et al. Dynamics of enterocyte tight junctions: effect of experimental colitis and two different anti-TNF strategies. Am J Physiol Gastrointest Liver Physiol. 2008;294:G938–47.

    Article  CAS  PubMed  Google Scholar 

  24. Suenaert P, Bulteel V, Lemmens L, et al. Anti-tumor necrosis factor treatment restores the gut barrier in Crohn’s disease. Am J Gastroenterol. 2002;97(8):2000–4.

    Article  CAS  PubMed  Google Scholar 

  25. Marchiando AM, Shen L, Graham WV, et al. Caveolin-1-dependent occludin endocytosis is required for TNF-induced tight junction regulation in vivo. J Cell Biol. 2010;189:111–26.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  26. Al-Sadi R, Boivin M, Ma T. Mechanism of cytokine modulation of epithelial tight junction barrier. Front Biosci Landmark Ed. 2009;14:2765–78.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  27. Shen L, Black ED, Witkowski ED, et al. Myosin light chain phosphorylation regulates barrier function by remodeling tight junction structure. J Cell Sci. 2006;119:2095–106.

    Article  CAS  PubMed  Google Scholar 

  28. Chen J, Zhang M. The Par-3/Par-6/aPKC complex and epithelial cell polarity. Exp Cell Res. 2013;319:1357–64.

    Article  CAS  PubMed  Google Scholar 

  29. Gopalakrishnan S, Hallett MA, Atkinson SJ, et al. aPKC-PAR complex dysfunction and tight junction disassembly in renal epithelial cells during ATP depletion. Am J Physiol Cell Physiol. 2007;292:C1094–102.

    Article  CAS  PubMed  Google Scholar 

  30. McCaffrey LM, Montalbano J, Mihai C, et al. Loss of the Par-3 polarity protein promotes breast tumorigenesis and metastasis. Cancer Cell. 2012;22:601–14.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  31. Nighot PK, Blikslager AT. Chloride channel ClC-2 modulates tight junction barrier function via intracellular trafficking of occludin. Am J Physiol Cell Physiol. 2012;302:C178–87.

    Article  CAS  PubMed  Google Scholar 

  32. Balda MS, Whitney JA, Flores C, et al. Functional dissociation of paracellular permeability and transepithelial electrical resistance and disruption of the apical-basolateral intramembrane diffusion barrier by expression of a mutant tight junction membrane protein. J Cell Biol. 1996;134:1031–49.

    Article  CAS  PubMed  Google Scholar 

  33. Yu AS, McCarthy KM, Francis SA, et al. Knockdown of occludin expression leads to diverse phenotypic alterations in epithelial cells. Am J Physiol Cell Physiol. 2005;288:C1231–41.

    Article  CAS  PubMed  Google Scholar 

  34. McCarthy KM, Skare IB, Stankewich MC, et al. Occludin is a functional component of the tight junction. J Cell Sci. 1996;109:2287–98.

    CAS  PubMed  Google Scholar 

  35. 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.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  36. Saitou M, Furuse M, Sasaki H, et al. Complex phenotype of mice lacking occludin, a component of tight junction strands. Mol Biol Cell. 2000;11:4131–42.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  37. Raleigh DR, Marchiando AM, Zhang Y, et al. Tight junction-associated MARVEL proteins marveld3, tricellulin, and occludin have distinct but overlapping functions. Mol Biol Cell. 2010;21:1200–13.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  38. Ivanov AI, Nusrat A, Parkos CA. Endocytosis of epithelial apical junctional proteins by a clathrin-mediated pathway into a unique storage compartment. Mol Biol Cell. 2004;15:176–88.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  39. Shen L, Turner JR. Actin depolymerization disrupts tight junctions via caveolae-mediated endocytosis. Mol Biol Cell. 2005;16:3919–36.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  40. Kienzle C, von Blume J. Secretory cargo sorting at the trans-Golgi network. Trends Cell Biol. 2014;24:584–93.

    Article  CAS  PubMed  Google Scholar 

  41. Wakana Y, van Galen J, Meissner F, et al. A new class of carriers that transport selective cargo from the trans Golgi network to the cell surface. EMBO J. 2012;31:3976–90.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  42. Jin Y, Atkinson SJ, Marrs JA, et al. Myosin II light chain phosphorylation regulates membrane localization and apoptotic signaling of tumor necrosis factor receptor-1. J Biol Chem. 2001;276:30342–9.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  43. Matsui T, Watanabe T, Matsuzawa K, et al. PAR-3 and aPKC regulate Golgi organization through CLASP2 phosphorylation to generate cell polarity. Mol Biol Cell. 2015;26:751–61.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  44. Odell AF, Hollstein M, Ponnambalam S, et al. A VE-cadherin–PAR-3–α-catenin complex regulates the Golgi localization and activity of cytosolic phospholipase A2α in endothelial cells. Mol Biol Cell. 2012;23:1783–96.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This research was supported by grants from the National Natural Science Foundation of China (NSFC 81330013 and NSFC 81272078 to H.Y., NSFC 81200288 to W.S.W., NSFC 81270451 to W.DX.), and the Program of Changjiang Scholars and Innovative Research (IRT 13050 to H.Y.).

Conflict of interest

The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. Department of General Surgery, Xinqiao Hospital, Third Military Medical University, Chongqing, 400037, China

    Min Yu, Songwei Yang, Yuan Qiu, Guoqing Chen, Wensheng Wang, Chao Xu, Lihua Sun, Weidong Xiao & Hua Yang

  2. Center of Medical Experiment and Technology, Xinqiao Hospital, Third Military Medical University, Chongqing, China

    Wenqiang Cai

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  1. Min Yu
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  2. Songwei Yang
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  3. Yuan Qiu
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Correspondence to Hua Yang.

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Yu, M., Yang, S., Qiu, Y. et al. Par-3 modulates intestinal epithelial barrier function through regulating intracellular trafficking of occludin and myosin light chain phosphorylation. J Gastroenterol 50, 1103–1113 (2015). https://doi.org/10.1007/s00535-015-1066-z

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  • DOI: https://doi.org/10.1007/s00535-015-1066-z

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