Caveolin is present in intestinal cells: role in cholesterol trafficking?
It was postulated that specialized microdomains of the plasma membrane, consistent with caveolae, might play a role in cholesterol trafficking in intestinal cells. The existence, therefore, of caveolin and the role of detergent-resistant microdomains of the plasma membrane in cholesterol trafficking were investigated in human small intestine and CaCo-2 cells. Caveolin mRNA was detected by RT-PCR in small intestinal brushings and biopsies and in CaCo-2 cells. Northern hybridization of caveolin mRNA detected 3 kb and 0.8 kb transcripts in CaCo-2 cells. From brushings of distal duodenum and in CaCo-2 cells, Western analysis for detection of caveolin protein demonstrated a 21 kDa-sized protein and a 600 kDa homooligomer. In CaCo-2 cells, caveolin was demonstrated by immunofluorescence in apical membranes as well as within cells. Using sucrose-density gradients, caveolin was localized to detergent-resistant microdomains of the plasma membrane. As determined by cholesterol oxidase-accessible cholesterol, 3-5% of plasma membrane cholesterol in CaCo-2 cells was estimated to be in these detergent-resistant microdomains. After the absorption of cholesterol from bile-salt micelles, more plasma membrane cholesterol moved to these specialized microdomains within the plasma membrane and was esterified. In CaCo-2 cells, filipin, N-ethyl maleimide, and cholesterol depletion, treatments that disrupt caveolar function, interfered with the transport of plasma membrane cholesterol to the endoplasmic reticulum, whereas okadaic acid, sphingomyelinase, and cholesterol oxidase did not. Changes in cholesterol flux at the apical membrane of the cell did not alter mRNA levels or mass of caveolin. The results suggest that caveolin is present in intestinal and CaCo-2 cells and is associated with detergent-resistant microdomains of cellular membranes. With the influx of micellar cholesterol from the lumen, plasma membrane cholesterol moves or "clusters" to these microdomains and is transported to the endoplasmic reticulum for esterification and eventual transport. Caveolin/caveolae may play a role in cholesterol trafficking in intestinal cells.
Field
F J
FJ
Department of Internal Medicine and the Veterans Administration, University of Iowa, Iowa City 52242, USA.
Born
E
E
Murthy
S
S
Mathur
S N
SN
D001692:Biological Transport; D001706:Biopsy; D015152:Blotting, Northern; D015153:Blotting, Western; D018938:Caco-2 Cells; D051242:Caveolin 1; D022461:Caveolins; D002462:Cell Membrane / Q000648:ultrastructure; D002784:Cholesterol / Q000378:metabolism*; D003902:Detergents; D004386:Duodenum / Q000737:chemistry / Q000648:ultrastructure; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D006801:Humans; D007422:Intestines / Q000737:chemistry*; D008565:Membrane Proteins / Q000032:analysis* / Q000235:genetics / Q000502:physiology*; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012995:Solubility
C495540:CAV1 protein, human; D051242:Caveolin 1; D022461:Caveolins; D003902:Detergents; D008565:Membrane Proteins; D012333:RNA, Messenger; D002784:Cholesterol