Caveolin-1 and Lipid Rafts in Confluent BeWo Trophoblasts: Evidence for Rock-1 Association with Caveolin-1

• Published in: Placenta (Eastbourne) 2007-02, Vol.28 (2), p.139-151
• Main Authors: Rashid-Doubell, F, Tannetta, D, Redman, C.W.G, Sargent, I.L, Boyd, C.A.R, Linton, E.A
• Format: Article
• Language: English
• Subjects: BeWo; Biological and medical sciences; Caveolin 1 - metabolism; Caveolin-1; Cell Differentiation; Cell Line, Tumor; Embryology: invertebrates and vertebrates. Teratology; Fluorescent Antibody Technique; Fundamental and applied biological sciences. Psychology; Gene Expression; Humans; Immunoprecipitation; Internal Medicine; Intracellular Signaling Peptides and Proteins - metabolism; Lipid rafts; Membrane Microdomains - metabolism; Microscopy, Confocal; Mitochondria; Mitochondria - metabolism; Obstetrics and Gynecology; Protein-Serine-Threonine Kinases - metabolism; rho-Associated Kinases; Rock-1; Syncytialisation; Trophoblasts - metabolism
• ISSN: 0143-4004; 1532-3102
• DOI: 10.1016/j.placenta.2005.12.005

Abstract Lipid rafts are detergent-insoluble, low-density membrane domains that are rich in cholesterol and sphingolipids; caveolae are a subdomain of the biochemically defined glycolipid raft whose expression is associated with the protein caveolin-1. This protein associates with numerous signalling molecules, regulating their activity by holding them inactive. Human villous cytotrophoblasts contain caveolin-1, but levels reduce greatly during their differentiation into syncytiotrophoblast. Since caveolin-1 is a known regulator of apoptosis and trophoblast syncytialisation involves the apoptotic cascade, we hypothesised that cytotrophoblast caveolin-1 may also play a role in regulating fusion events involved in syncytium formation. The BeWo choriocarcinoma cell line has previously proved valuable for studying trophoblast syncytialisation, hence the present work was carried out to determine whether BeWo cells could be used as a model for the exploration of caveolin-1's role in regulating the syncytialisation process. Undifferentiated BeWo cells were found to express caveolin-1 in similar amounts to villous cytotrophoblasts isolated from term placenta. Lipid raft fractions prepared from these BeWo cells at confluence contained the raft-associated proteins caveolin-1 and -2, flotillin-1 and -2, stomatin and the heterotrimeric G protein, Gαq. Confocal immunofluorescence studies revealed that caveolin-1 is internalized to the mitochondria, but not to the Golgi or endoplasmic reticulum, in subconfluent BeWo and that the protein relocates to the plasma membrane upon confluence, an observation confirmed by caveolin-1 and cytochrome c Western blotting of lipid raft fractions and mitochondria purified from confluent and subconfluent cells. Western blotting and immunofluorescence experiments comparing undifferentiated cells and those induced to differentiate using the cAMP analogue, dibutyryl cAMP, showed that BeWo syncytialisation was accompanied by a reduction in caveolin-1 levels, similar to the situation in primary villous cytotrophoblasts. Confluent, undifferentiated BeWo cultures were then used to investigate the cellular localisation of Rock-1, a protein which promotes cytoskeletal re-organisation important for syncytialisation and apoptosis. Its association with caveolin-1 was evidenced by the demonstration that the 160 kDa proenzyme form of Rock-1 co-immunoprecipitates with caveolin-1 and vice versa, as well as by the co-localisation of the two proteins at