Golgi fragmentation is associated with ceramide-induced cellular effects.
Ceramide has been shown to cause anoikis, a subtype of apoptosis due to inadequate cell adhesion. However, the underlying mechanism is unclear. Herein, we report that D-e-C6-ceramide (D-e-Cer), via generating sphingosine, disrupts the Golgi complex (GC), which is associated with various cellular effects, including anoikis. Treatment of HeLa cells with D-e-Cer caused cell elongation, spreading inhibition, rounding, and detachment before apoptosis (anoikis). In D-e-Cer-treated cells, glycosylation of beta1 integrin in the GC was inhibited, thus its associated integrin receptors failed to translocate to the cell surface. Ceramide treatment also inhibited the reorganization of both microtubule and F-actin cytoskeletons, focal adhesions, and filopodia. These cellular effects were preceded by fragmentation of the Golgi complex. In contrast, L-e-C6-ceramide (L-e-Cer), the enantiomer of D-e-Cer, failed to induce these cellular effects. Mass spectrometric analysis revealed that treatment HeLa cells with D-e-Cer but not L-e-Cer caused a >50-fold increase in the levels of sphingosine, a product of hydrolysis of ceramide. Treatment with D-e-sphingosine and its enantiomer, L-e-sphingosine, caused massive perinuclear vacuolization, Golgi fragmentation, and cell rounding. Together, these results suggest that sphingosine generated from hydrolysis of ceramide causes the GC disruption, leading to various cellular effects.
Hu
Wei
W
Department of Medicine and Biochemistry, Medical University of South Carolina, Charleston, SC 28425, USA.
Xu
Ruijuan
R
Zhang
Guofeng
G
Jin
Junfei
J
Szulc
Zdzislaw M
ZM
Bielawski
Jacek
J
Hannun
Yusuf A
YA
Obeid
Lina M
LM
Mao
Cungui
C
D000199:Actins / Q000737:chemistry / Q000378:metabolism; D023102:Anoikis; D017209:Apoptosis; D019921:Biotinylation; D015153:Blotting, Western; D020126:Brefeldin A / Q000494:pharmacology; D002448:Cell Adhesion; D002453:Cell Cycle; D002462:Cell Membrane / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D049109:Cell Proliferation; D002469:Cell Separation; D002470:Cell Survival; D002518:Ceramides / Q000378:metabolism / Q000494:pharmacology*; D003599:Cytoskeleton / Q000737:chemistry / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005434:Flow Cytometry; D006031:Glycosylation; D006056:Golgi Apparatus / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D006868:Hydrolysis; D019012:Integrin beta1 / Q000378:metabolism; D018613:Microscopy, Confocal; D008854:Microscopy, Electron; D008870:Microtubules / Q000378:metabolism; D008954:Models, Biological; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D021381:Protein Transport; D011554:Pseudopodia / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D013110:Sphingosine / Q000737:chemistry / Q000494:pharmacology; D013237:Stereoisomerism; D013997:Time Factors; D014162:Transfection
D000199:Actins; D002518:Ceramides; D019012:Integrin beta1; D010455:Peptides; C101954:N-caproylsphingosine; D020126:Brefeldin A; D013110:Sphingosine