Cholesterol is required for the polarized secretion of erythropoietin in Madin-Darby canine kidney cells.
It has already been reported that stably expressed exogenous human wild-type EPO (wtEPO) is preferentially secreted to the apical side and one of the three N-linked carbohydrate chains critically acts as an apical sorting determinant in Madin-Darby canine kidney (MDCK) cells. It has been suggested that lipid rafts are involved in the apical sorting of membrane and secretory proteins. To investigate the involvement of lipid rafts in the apical sorting of wtEPO, we examined the effect of cholesterol depletion with methyl-beta-cyclodextrin on the secretion polarity of EPO and analyzed Triton X-100 insoluble cell extracts by sucrose density gradients centrifugation in MDCK cells. We found that wtEPO was shifted in non-polarized direction by cholesterol depletion. Most of the wtEPO was not detectable in the raft fractions by sucrose density gradients centrifugation analysis. These results indicate that apical secretion of EPO involves a cholesterol-dependent mechanism probably not involving lipid rafts.
Maruyama
Masato
M
Department of Biopharmaceutics and Drug Metabolism, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Japan.
Kishimoto
Michiyuki
M
Ishida
Kayo
K
Watanabe
Yoshihiko
Y
Nishikawa
Makiya
M
Masuda
Seiji
S
Sasaki
Ryuzo
R
Takakura
Yoshinobu
Y
D000818:Animals; D001681:Biological Assay; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D002784:Cholesterol / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D003902:Detergents / Q000494:pharmacology; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D004921:Erythropoietin / Q000235:genetics / Q000378:metabolism*; D015151:Immunoblotting; D016898:Interferon-alpha / Q000378:metabolism; D016899:Interferon-beta / Q000378:metabolism; D007477:Ions; D007668:Kidney / Q000166:cytology*; D021962:Membrane Microdomains / Q000378:metabolism; D009154:Mutation; D017830:Octoxynol / Q000494:pharmacology; D010957:Plasmids / Q000378:metabolism; D013395:Sucrose / Q000737:chemistry / Q000494:pharmacology; D013997:Time Factors; D014162:Transfection
D003902:Detergents; D016898:Interferon-alpha; D007477:Ions; D004921:Erythropoietin; D013395:Sucrose; D016899:Interferon-beta; D017830:Octoxynol; D002784:Cholesterol