Effects of methyl-beta-cyclodextrin treatment on secretion profile of interferon-beta and zonula occuludin-1 architecture in Madin-Darby canine kidney cell monolayers.
The interferon (IFN) is a paradigm of secretory protein. However, it has been poorly understood how its secretion is regulated in polarized epithelial cells. Recently, we had shown that exogenous IFNs transiently expressed in polarized monolayers were predominantly secreted to the side on which gene transfection had been performed, while stably expressed IFNs were secreted almost equally to the both cell sides. Since those modes of secretion did not affect each other, epithelial cell layers seemed to have at least two protein sorting/secretion pathways, one for transient expression and the other for stable expression, for identical secretory proteins. Furthermore, this dual secretion profile seemed to be mediated by distinct post-trans Golgi network vesicles, suggesting the involvement of lipid rafts in the sorting multiplicity. To address this issue, here we studied the effects of cholesterol depletion with methyl-beta-cyclodextrin (MbetaCD) on the secretion profile of IFN-beta exogenously expressed in Madin-Darby canine kidney (MDCK) cells. The MbetaCD-treatment, however, did not affect the profile in either transient or stable expression, although the architecture of zonula occuludin-1, which links to the tight junction, was substantially disrupted by the treatment. Further analysis of Triton X-100-insoluble cell extracts by sucrose density centrifugation demonstrated that IFN-beta was not apparently associated with lipid rafts in either transient or stable expression. These results suggest that lipid rafts may not be crucially involved in the regulation of secretion polarity of IFN-beta in the epithelial cells.
Department of Biopharmaceutics and Drug Metabolism, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan.
Masato
M
Maruyama
Kayo
K
Ishida
Yoshihiko
Y
Watanabe
Makiya
M
Nishikawa
Yoshinobu
Y
Takakura
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D016764:Cell Polarity / Q000187:drug effects; D002784:Cholesterol / Q000378:metabolism; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D006801:Humans; D016899:Interferon-beta / Q000235:genetics* / Q000378:metabolism*; D007668:Kidney / Q000166:cytology*; D021962:Membrane Microdomains / Q000187:drug effects / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D018613:Microscopy, Confocal; D010750:Phosphoproteins / Q000378:metabolism*; D010957:Plasmids; D019108:Tight Junctions / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D014162:Transfection; D062826:Zonula Occludens-1 Protein; D047392:beta-Cyclodextrins / Q000494:pharmacology*
D008565:Membrane Proteins; D010750:Phosphoproteins; C569042:TJP1 protein, human; C569043:Tjp1 protein, mouse; D062826:Zonula Occludens-1 Protein; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D016899:Interferon-beta; D002784:Cholesterol