, Volume 31, Issue 8, pp 817–828 | Cite as

Alterations in cell cholesterol content modulate Ca2+-induced tight junction assembly by MDCK cells

  • Michael C. Stankewich
  • Stacy A. Francis
  • Quynh U. Vu
  • Eveline E. Schneeberger
  • Robert D. Lynch


Transepithelial electrical resistance (TER), a measure of tight junction (TJ) barrier function, develops more rapidly and reaches higher values after preincubation of MDCK cells for 24 h with 2 μM Lovastatin (lova), an inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase. While this effect was attributed to a 30% fall in cholesterol (CH), possible effects of lova on the supply of prenyl group precursors could not be excluded. In the current study, strategies were devised to examine effects on TER of agents that simultaneously lower CH and increase the flux of intermediates through the CH biosynthetic pathway. Zaragozic acid, 20 μM, an inhibitor of squalene synthase known to increase the synthesis of isoprenoids and levels of prenylated proteins, lowered cell CH by 30% after 24 h, while accelerating development of TER in the same manner as lova. TER was also enhanced, despite a 23% increase in the rate of [3H]acetate incorporation into CH, when total CH was reduced by 45% during a 2-h incubation with 2 mM methyl β-cyclodextrin (MBCD), an agent that stimulates CH efflux from cells. The fact that the rate of TER development was diminished when cell CH content was elevated by incubation with a complex of CH and MBCD is further evidence that this sterol modulates development of the epithelial barrier. Cell associated CH derived from the complex was similar to endogenous CH with respect to its accessibility to cholesterol oxidase. Lova's effect on TER was diminished when 5 μg/mL of CH was added to the medium during the last 11 h of incubation with lova.


Tight Junction Lovastatin MDCK Cell Transepithelial Electrical Resis Bovine Calf Serum 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



bovine calf serum




cholesterol-methyl β-cyclodextrin


Dulbecco's modified Eagle's medium


Earle's balanced salt solution






methyl β-cyclodextrin


phosphate-buffered saline




specific activity


trichloroacetic acid


transepithelial electrical resistance


tight junction


thin-layer chromatography


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Schneeberger, E.E., and Lynch, R.D. (1992) Structure, Function and Regulation of Cellular Tight Junctions,Am. J. Physiol. 262, L647-L661.PubMedGoogle Scholar
  2. 2.
    Furuse, M., Hirase, T., Itoh, M., Nagafuchi, A., Yonemura, S., Tsukita, S., and Tsukita, S. (1993) Occludin: A Novel Integral Membrane Protein Localizing at Tight Junctions,J. Cell Biol. 123, 1777–1788.PubMedCrossRefGoogle Scholar
  3. 3.
    Van Venetie, R., and Verkleij, A.J. (1981) Analysis of the Hexagonal II Phase and Its Relations to Lipidic Particles and the Lamellar Phase. A Freeze-Fracture Study,Biochim. Biophys. Acta 645, 262–269.PubMedCrossRefGoogle Scholar
  4. 4.
    Pinto da Silva, P., and Kachar, B. (1982) On Tight Junction Structure,Cell 28, 441–450.PubMedCrossRefGoogle Scholar
  5. 5.
    Kachar, B., and Reese, T.S. (1982) Evidence for the Lipidic Nature of Tight Junction Strands,Nature 296, 464–466.PubMedCrossRefGoogle Scholar
  6. 6.
    Turin, L., Behe, P., Plonsky, I., and Dunina-Barkovskaya, A. (1991) Hydrophobic Ion Transfer Between Membranes of Adjacent Hepatocytes: A Possible Probe of Tight Junction Structure,Proc. Natl. Acad. Sci. USA 88, 9365–9369.PubMedCrossRefGoogle Scholar
  7. 7.
    Grebenkamper, K., and Galla, H.J. (1994) Translational Diffusion Measurements of a Fluorescent Phospholipid Between MDCK I Cells Support the Lipid Model of the Tight Junctions,Chemistry & Physics of Lipids 71, 133–143.CrossRefGoogle Scholar
  8. 8.
    Yeagle, P.L. (1985) Cholesterol and the Cell Membrane,Biochim. Biophys. Acta 822, 267–287.PubMedGoogle Scholar
  9. 9.
    Yeagle, P.L., Young, J., and Rice, D. (1988) Effects of Cholesterol On Na+,K+-ATPase ATP Hydrolyzing Activity in Bovine Kidney,Biochem. 27, 6449–6452.CrossRefGoogle Scholar
  10. 10.
    Chernomordik, L., Kozlov, M.M., and Zimmerberg, J. (1995) Lipids in Biological Membrane Fusion,J. Membr. Biol. 146, 1–14.PubMedGoogle Scholar
  11. 11.
    Balda, M.S., Gonzalez-Mariscal, L., Contreras, R.G., Macias-Silva, M., Torres-Marquez, M.E., Garcia-Sainz, J.A., and Cereijido, M. (1991) Assembly and Sealing of Tight Junctions: Possible Participation of G-Proteins, Phospholipase C, Protein Kinase C and Calmodulin,J. Membr. Biol. 122, 193–202.PubMedCrossRefGoogle Scholar
  12. 12.
    Schneeberger, E.E., Lynch, R.D., Kelly, C.A., and Rabito, C.A. (1988) Modulation of Tight Junction Formation in Clone 4 MDCK Cells by Fatty Acid Supplementation,Am. J. Physiol. 254, C432-C440.PubMedGoogle Scholar
  13. 13.
    Lazaro, A., Calderon, V., Gonzalez-Mariscal, L., Contrereas, R.G., Valdez, J., Zampighi, G., and Cereijido, M. (1991) Tight Junctions, Lipids, and Transepithelial Electrical Resistance (TER),J. Cell Biol., 115, 481a.Google Scholar
  14. 14.
    Lange, Y., Swaisgood, M.H., Ramos, B.V., and Steck, T.L. (1989) Plasma Membranes Contain Half the Phospholipid and 90% of the Cholesterol and Sphingomyelin in Cultured Human Fibroblasts,J. Biol. Chem. 264, 3786–3793.PubMedGoogle Scholar
  15. 15.
    Yeagle, P. (1987)The Membranes of Cells, p. 122. Academic Press, Inc., New York.Google Scholar
  16. 16.
    Lynch, R.D., Tkachuk, L.J., Ji, X., Rabito, C.A., and Schneeberger, E.E. (1993) Depleting Cell Cholesterol Alters Calcium-Induced Assembly of Tight Junctions By Monolayers of MDCK Cells,Europ. J. Cell Biol. 60, 21–30.PubMedGoogle Scholar
  17. 17.
    Zahraoui, A., Joberty, G., Arpin, M., Fontaine, J.J., Hellio, R., Tavitian, A., and Louvard, D. (1994) A Small Rab GTPase Is Distributed in Cytoplasmic Vesicles in Nonpolarized Cells But Colocalizes With the TJ Marker ZO-1 in Polarized Epithelial Cells,J. Cell Biol. 124, 101–115.PubMedCrossRefGoogle Scholar
  18. 18.
    Repko, E.M., and Maltese, W.A. (1989) Post-Translational Isoprenylation of Cellular Proteins Is Altered in Response to Mevalonate Availability,J. Biol. Chem. 264, 9945–9952.PubMedGoogle Scholar
  19. 19.
    Beck, L.A., Hosick, T.J., and Sinensky, M. (1990) Isoprenylation Is Required for the Processing of the Lamin A Precursor,J. Cell Biol. 110, 148914–99.CrossRefGoogle Scholar
  20. 20.
    Fenton, R.G., Kung, H.-F., Longo, D.L., and Smith, M.R. (1992) Regulation of Intracellular Actin. Polymerization By Prenylated Cellular Proteins,J. Cell Biol. 117, 347–356.PubMedCrossRefGoogle Scholar
  21. 21.
    Ness, G.C., Zhao, Z., and Keller, R.K. (1994) Effect of Squalene Synthase Inhibition On the Expression of Hepatic Cholesterol Biosynthetic Enzymes, LDL Receptor, and Cholesterol 7 Alpha Hydroxylase,Archives of Biochemistry & Biophysics 311, 277–285.CrossRefGoogle Scholar
  22. 22.
    Correll, C.C., and Edwards, P.A. (1994) Mevalonic Acid-Dependent Degradation of 3-Hydroxy-3-Methylglutaryl-Coenzyme A Reductasein vivo andin vitro, J. Biol. Chem. 269, 633–638.PubMedGoogle Scholar
  23. 23.
    Gonzalez-Mariscal, L., Chavez De Ramirez, G., and Cereijido, M. (1985) Tight Junction Formation in Cultured Epithelial Cells (MDCK),J. Membr. Biol. 86, 113–125.PubMedCrossRefGoogle Scholar
  24. 24.
    Lynch, R.D., Tkachuk, L.J., McCormack, J.M., McCarthy, K.M., Rogers, R.A., and Schneeberger E.E. (1995) Basolateral But Not Apical Application of Protease Results in a Rapid Rise of Transepithelial Electrical Resistance and Formation of Aberrant Tight Junction Strands in MDCK Cells,Europ. J. Cell Biol. 66, 257–267.PubMedGoogle Scholar
  25. 25.
    Sigma Chemical Company (1992)Water-Soluble Complexes. Parts 1 & 2: Cyclodextrins in Cell Culture, Vol. 8, no. 1.Google Scholar
  26. 26.
    De Caprio, J., Yun, J., and Javitt, N.B. (1992) Bile Acid and Sterol Solubilization in 2-Hydroxypropyl-Beta-Cyclodextrin,J. Lipid Res. 33, 441–443.PubMedGoogle Scholar
  27. 27.
    Szejtli, J. (1988)Cyclodextrin Technology, pp. 79–109, Kluwer Academic Publishers, Dordrecht-Boston-London.Google Scholar
  28. 28.
    Petrack, B., and Latario, B.J. (1993) Synthesis of 27-Hydroxyc-holesterol in Rat Liver Mitochondria: HPLC Assay and Marked Deactivation By Exogenous Cholesterol,J. Lipid Res. 34, 643–649.PubMedGoogle Scholar
  29. 29.
    El Yandouzi, E.H., Zlatkine, P., Moll G., and Le Grimellec, C. (1994) Cholesterol Distribution in Renal Epithelial Cells LLC-PK1 As Determined By Cholesterol Oxidase: Evidence That Glutaraldehyde Fixation Masks Plasma Membrane Cholesterol Pools,Biochem. 33, 2329–2334.CrossRefGoogle Scholar
  30. 30.
    Goh, E.H., Krauth, D.K., and Colles, S.M. (1990) Analysis of Cholesterol and Desmosterol in Cultured Cells Without Organic Solvent Extraction,Lipids 25, 738–741.PubMedGoogle Scholar
  31. 31.
    Eastman Kodak (1978) U.S. Patent 4,093,517.Google Scholar
  32. 32.
    Bergstrom, J.D., Kurtz, M.M., Rew, D.J., Amend, A.M., Karkas, J.D., Bostedor, R.G., Bansal, V.S., Dufrensne, C., VanMiddlesworth, F.L., Hensens, O.D., Liesch, J.M., Zink, D.L., Wilson, K.E., Onishi, J., Milligan, J.A., Bills, G., Kaplan, L., Omstead, M.N., Jenkins, R.G., Huang, L., Meinz, M.S., Quinn, L., Burg, R.W., Kong, Y., Mochales, S., Mojena, M., Martin, I., Pelaez, F., Diez, M.T., and Alberts, A.W. (1993) Zaragozic Acids: A Family of Fungal Metabolites That Are Picomolar Competitive Inhibitors of Squalene Synthase,Proc. Natl. Acad. Sci. USA 90, 80–84.PubMedCrossRefGoogle Scholar
  33. 33.
    Nakanishi, M., Goldstein, J.L., and Brown, M.S. (1988) Multivalent Control of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase. Mevalonate-Derived Product Inhibits Translation of mRNA and Accelerates Degradation of Enzyme,J. Biol. Chem. 263, 8929–8937.PubMedGoogle Scholar
  34. 34.
    Chin, D.J., Gil, G.G., Goldstein, J.L., Brown, M.S., and Luskey, K.L. (1985) Sterols Accelerate Degradation of Hamster 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Encoded by a Constitutively Expressed cDNA,Molecular and Cellular Biology 5, 634–641.PubMedGoogle Scholar
  35. 35.
    Goldstein, J.L., and Brown, M.S. (1990) Regulation of the Mevalonate Pathway,Nature 343, 425–430.PubMedCrossRefGoogle Scholar
  36. 36.
    O'Donnell, M.P., Kasiske, B.L., Kim, Y., Atluru, D., and Keane, W.F. (1993) Lovastatin Inhibits Proliferation of Rat Mesangial Cells,J. Clin. Invest. 91, 83–87.PubMedCrossRefGoogle Scholar
  37. 37.
    López-Nicolás, J., Bru, R., Sánchez-Ferrer, A., and García-Carmona, F. (1995) Use of “Soluble Lipids” for Biochemical Processes: Linoleic Acid-Cyclodextrin Inclusion Complexes in Aqueous Solutions,Biochem. J. 308, 151–154.PubMedGoogle Scholar
  38. 38.
    Kilsdonk, E.P.C., Yancey, P.G., Stoudt, G.W., Bangeter, F.W., Johnson, W.J., Phillips, M.C., and Rothblatt, G.H. (1995) Cellular Cholesterol Efflux Mediated By Cyclodextrins,J. Biol. Chem. 270, 17250–17256.PubMedCrossRefGoogle Scholar
  39. 39.
    Hidaka, Y., Hotta, H., Nagata, Y., Iwasawa, Y., Horie, M., and Kamei, T. (1991) Effect of a Novel Squalene Epoxidase Inhibitor, NB-598, On the Regulation of Cholesterol Metabolism in Hep G2 Cells,J. Biol. Chem. 266, 13171–13177.PubMedGoogle Scholar
  40. 40.
    Kam, N.T.P., Albright, E., Mathur, S., and Field, F.J. (1990) Effect of Lovastatin on Acyl-CoA: Cholesterol O-Acyltransferase (ACAT) Activity and the Basolateral-Membrane Secretion of Newly Synthesized Lipids by CaCo2 Cells,Biochem. J. 272, 427–433.PubMedGoogle Scholar
  41. 41.
    Porn, M.I., and Slotte, J.P. (1995) Localization of Cholesterol in Sphingomyelinase-Treated Fibroblasts,Biochem. J. 308, 269–274.PubMedGoogle Scholar
  42. 42.
    Smart, E.J., Ying, Y.S., Conrad, P.A., and Anderson, R.G. (1994) Caveolin Moves from Caveolae to the Golgi Apparatus in Response to Cholesterol Oxidation,J. Cell Biol. 127, 1185–1197.PubMedCrossRefGoogle Scholar
  43. 43.
    Hoover, R.L., Dawidowicz, E.A., Robinson, J.M., and Karnovsky, M.J. (1983) Role of Cholesterol in the Capping of Surface Immunoglobulin Receptors On Murine Lymphocytes,J. Cell Biol. 97, 73–80.PubMedCrossRefGoogle Scholar
  44. 44.
    Eklund, K.K., Takkunen, J.E., and Kinnunen, P.K. (1991) Cation-Induced Aggregation of Acidic Phospholipid Vesicles: The Role of Fatty Acid Unsaturation and Cholesterol,Chemistry & Physics of Lipids, 57, 59–66.CrossRefGoogle Scholar
  45. 45.
    Parasassi, T., Di Stefano, M., Loiero, M., Ravagnan, G., and Gratton, E. (1994) Influence of Cholesterol On Phospholipid Bilayers Phase Domains As Detected By Laurdan Fluorescence,Biophysical J. 66, 120–132.CrossRefGoogle Scholar
  46. 46.
    Yeagle, P.L. (1989) Lipid Regulation of Cell Membrane Structure and Function,FASEB Journal 3, 1833–1842.PubMedGoogle Scholar
  47. 47.
    Contreras, R.G., Miller, J.H., Zamora, M., Gonzalez-Mariscal, L., and Cereijido, M. (1992) Interaction of Calcium with Plasma Membrane of Epithelial (MDCK) Cells During Junction Formation,Am. J. Physiol., 263, C313-C318.PubMedGoogle Scholar
  48. 48.
    Balda, M.S., Gonzalez-Mariscal, L., Matter, K., Cereijido, M., and Anderson, J.M. (1993) Assembly of the Tight Junction: The Role of Diacylglycerol,J. Cell Biol. 123, 293–302.PubMedCrossRefGoogle Scholar
  49. 49.
    Traub, P., Perides, G., Kuhn, S., and Scherbarth, A. (1987) Efficient Interaction of Nonpolar Lipids with Intermediate Filaments of the Vimentin Type,Europ. J. Cell Biol. 43, 55–64.PubMedGoogle Scholar
  50. 50.
    Perides, G., Harter, C., and Traub, P. (1987) Electrostatic and Hydrophobic Interactions of the Intermediate Filament Protein Vimentin and Its Amino Terminus with Lipid Bilayers,J. Biol. Chem. 262, 13742–13749.PubMedGoogle Scholar
  51. 51.
    Chen, M., Mason, R.P., and Tulenko, T.N. (1995) Atherosclerosis Alters the Composition, Structure and Function of Arterial Smooth Muscle Cell Plasma Membranes,Biochim. Biophys. Acta 1272, 101–112.PubMedGoogle Scholar

Copyright information

© AOCS Press 1996

Authors and Affiliations

  • Michael C. Stankewich
    • 1
  • Stacy A. Francis
    • 1
  • Quynh U. Vu
    • 1
  • Eveline E. Schneeberger
    • 2
  • Robert D. Lynch
    • 1
  1. 1.Department of Biological SciencesUniversity of Massachusetts at LowellLowell
  2. 2.Department of PathologyMassachusetts General HospitalBoston

Personalised recommendations