Caveolin isoform expression during differentiation of C6 glioma cells.
Caveolae, a specialized form of lipid rafts, are cholesterol- and sphingolipid-rich membrane microdomains implicated in potocytosis, endocytosis, transcytosis, and as platforms for signal transduction. One of the major constituents of caveolae are three highly homologous caveolin isoforms (caveolin-1, caveolin-2, and caveolin-3). The present study expands the analysis of caveolin isoform expression in C6 glioma cells. Three complementary approaches were used to assess their differential expression during the dibutyryl-cyclic AMP-induced differentiation of C6 cells into an astrocyte-like phenotype. Immunoblotting, conventional RT-PCR, and real-time RT-PCR analysis established the expression of the caveolin-3 isoform in C6 cells, in addition to caveolin-1 and caveolin-2. Similar to the other isoforms, caveolin-3 was associated with light-density, detergent-insoluble caveolae membrane fractions obtained using sucrose-density gradient centrifugation. The three caveolin isoforms display different temporal patterns of mRNA/protein expression during the differentiation of C6 cells. Western blot and real-time RT-PCR analysis demonstrate that caveolin-1 and caveolin-2 are up-regulated during the late stages of the differentiation of C6 cells. Meanwhile, caveolin-3 is gradually down-regulated during the differentiation process. Indirect immunofluorescence analysis via laser-scanning confocal microscopy reveals that the three caveolin isoforms display similar subcellular distribution patterns. In addition, co-localization of caveolin-1/caveolin-2 and caveolin-1/caveolin-3 was detected in both C6 glioma phenotypes. The findings reveal a differential temporal pattern of caveolin gene expression during phenotypic differentiation of C6 glioma cells, with potential implications to developmental and degenerative events in the brain.
Silva
W I
WI
Department of Physiology, School of Medicine, University of Puerto Rico, Medical Sciences Campus, P.O. Box 365067, San Juan, PR. wsilva@rcm.upr.edu
Maldonado
H M
HM
Velázquez
G
G
Rubio-Dávila
M
M
Miranda
J D
JD
Aquino
E
E
Mayol
N
N
Cruz-Torres
A
A
Jardón
J
J
Salgado-Villanueva
I K
IK
D000818:Animals; D001253:Astrocytes / Q000378:metabolism; D015153:Blotting, Western; D051242:Caveolin 1 / Q000096:biosynthesis* / Q000737:chemistry; D002454:Cell Differentiation / Q000502:physiology; D002458:Cell Fractionation; D045744:Cell Line, Tumor; D002499:Centrifugation, Density Gradient; D017931:DNA Primers; D015536:Down-Regulation; D004591:Electrophoresis, Polyacrylamide Gel; D019084:Fluorescent Antibody Technique, Indirect; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D005910:Glioma / Q000378:metabolism*; D007536:Isomerism; D018613:Microscopy, Confocal; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction
C495537:Cav1 protein, rat; D051242:Caveolin 1; D017931:DNA Primers; D005904:Glial Fibrillary Acidic Protein; D012333:RNA, Messenger