Ocadaic acid treatment alters the intracellular localization of caveolin-1 and caveolin-2 in HepG2 cells

In this paper we provide evidences that protein phosphatases could regulate the intracellular localization of caveolin isoforms in a hepatoma cell line (HepG2). Ocadaic acid (OA) - a serine/threonine phosphatase inhibitor – was used in various concentrations (4nM and 100nM) to study the localization of caveolin-1 and caveolin-2 in HepG2 cells. Using fluorescent and confocal immunocytochemistry we have found that OA in both concentrations has significantly altered the intracellular localization and distribution of the caveolin-1 and caveolin-2 as well. In control (-OA treatment) the caveolin-1 was present in discrete punctate structures in the cytoplasm and also on the cell membrane. Caveolin-2 has partly overlapped with caveolin-1, but a significant amount caveolin-2 was detected around the nucleus. After OA (4 and 100 nM) treatment caveolin-1 has disappeared from the cell membrane, it was present mainly in the cytoplasm in larger vesicle or vacuole-like structures that were arranged along the cables of the cytoskeleton. In many cases caveolin-2 was found to colocalize with caveolin-1, but there was always a significant amount of caveolin-2 present around the nucleus. Immunoprecipitation and Western blot analysis revealed that in OA-treated cells a ~24 kDa protein identified as caveolin-2 was strongly phosphorylated on tyrosine residues. The effect of OA was not reversible, since the removal of OA has not resulted in the dephosphorylation of caveolin-2 and the perinuclear localization of caveolin-2 remained. Our data indicate that phophorylation of caveolin-2 can alter not only the intracellular localization of caveolin isoforms but also the distribution of caveolae. The cytoskeleton seems to play an important role in the normal and altered distribution of caveolae, and the tyrosine phosphorylation or the absence of dephosphorylation of caveolin-2 isoform can inhibit the recycling of caveolae

Caveolin-2 associates with intracellular chlamydial inclusions independently of caveolin-1

BACKGROUND: Lipid raft domains form in plasma membranes of eukaryotic cells by the tight packing of glycosphingolipids and cholesterol. Caveolae are invaginated structures that form in lipid raft domains when the protein caveolin-1 is expressed. The Chlamydiaceae are obligate intracellular bacterial pathogens that replicate entirely within inclusions that develop from the phagocytic vacuoles in which they enter. We recently found that host cell caveolin-1 is associated with the intracellular vacuoles and inclusions of some chlamydial strains and species, and that entry of those strains depends on intact lipid raft domains. Caveolin-2 is another member of the caveolin family of proteins that is present in caveolae, but of unknown function. METHODS: We utilized a caveolin-1 negative/caveolin-2 positive FRT cell line and laser confocal immunofluorescence techniques to visualize the colocalization of caveolin-2 with the chlamydial inclusions. RESULTS: We show here that in infected HeLa cells, caveolin-2, as well as caveolin-1, colocalizes with inclusions of C. pneumoniae (Cp), C. caviae (GPIC), and C. trachomatis serovars E, F and K. In addition, caveolin-2 also associates with C. trachomatis serovars A, B and C, although caveolin-1 did not colocalize with these organisms. Moreover, caveolin-2 appears to be specifically, or indirectly, associated with the pathogens at the inclusion membranes. Using caveolin-1 deficient FRT cells, we show that although caveolin-2 normally is not transported out of the Golgi in the absence of caveolin-1, it nevertheless colocalizes with chlamydial inclusions in these cells. However, our results also show that caveolin-2 did not colocalize with UV-irradiated Chlamydia in FRT cells, suggesting that in these caveolin-1 negative cells, pathogen viability and very likely pathogen gene expression are necessary for the acquisition of caveolin-2 from the Golgi. CONCLUSION: Caveolin-2 associates with the chlamydial inclusion independently of caveolin-1. The function of caveolin-2, either in the uninfected cell or in the chlamydial developmental cycle, remains to be elucidated. Nevertheless, this second caveolin protein can now be added to the small number of host proteins that are associated with the inclusions of this obligate intracellular pathogen

Sterol Carrier Protein-2 Directly Interacts with Caveolin-1 in Vitro and in Vivo

HDL-mediated reverse-cholesterol transport as well as phosphoinositide signaling are mediated through plasma membrane microdomains termed caveolae/lipid rafts. However, relatively little is known regarding mechanism(s) whereby these lipids traffic to or are targeted to caveolae/lipid rafts. Since sterol carrier protein-2 (SCP-2) binds both cholesterol and phosphatidylinositol, the possibility that SCP-2 might interact with caveolin-1 and caveolae was examined. Double immunolabeling and laser scanning fluorescence microscopy showed that a small but significant portion of SCP-2 colocalized with caveolin-1 primarily at the plasma membrane of L-cells and more so within intracellular punctuate structures in hepatoma cells. In SCP-2 overexpressing L-cells, SCP-2 was detected in close proximity to caveolin, 48 ± 4 Å, as determined by fluorescence resonance energy transfer (FRET) and immunogold electron microscopy. Cell fractionation of SCP-2 overexpressing L-cells and Western blotting detected SCP-2 in purified plasma membranes, especially in caveolae/ lipid rafts as compared to the nonraft fraction. SCP-2 and caveolin-1 were coimmunoprecipitated from cell lysates by anti-caveolin-1 and anti-SCP-2. Finally, a yeast two-hybrid assay demonstrated that SCP-2 directly interacts with caveolin-1 in vivo. These interactions of SCP-2 with caveolin-1 were specific since a functionally related protein, phosphatidyinositol transfer protein (PITP), colocalized much less well with caveolin-1, was not in close proximity to caveolin-1 (i.e., \u3e120 Å), and was not coimmunoprecipitated by anti-caveolin-1 from cell lysates. In summary, it was shown for the first time that SCP-2 (but not PITP) selectively interacted with caveolin-1, both within the cytoplasm and at the plasma membrane. These data contribute significantly to our understanding of the role of SCP-2 in cholesterol and phosphatidylinositol targeted from intracellular sites of synthesis in the endoplasmic reticulum to caveolae/lipid rafts at the cell surface plasma membrane

Identifying the role of caveolin-1 and caveolin-2 in cancer cell proliferation and cancer drug resistance [abstract]

Abstract only availableFaculty Mentor: Dr. Grzegorz Sowa, Medical Pharmacology and PhysiologyCaveolae are flask-shaped invaginations of the plasma membrane. Caveolins are the major protein component building these caveolae. Caveolins are involved in cell signaling and play a role multiple subcellular processes, such as endocytosis. Of the three types of caveolins (numbered 1, 2, and 3), most research has been performed on caveolin-1. For example, in the field of cancer, it has been found that caveolin-1 has the ability to either suppress or encourage growth of cancer cells (depending on the type of cancer). Interestingly, it has been reported that the level of both caveolin-1 and -2 increases in drug resistant cancer cell lines. However, it is unknown if such increases are coincidental or actually play an important role in the development of drug resistance. The major goal of this study is to better understand the role of caveolins-1 and -2 in the growth and drug resistance of A549 lung adenocarcinoma. For this purpose, the levels of caveolin-1 and -2 were dramatically reduced (knocked down) using small interfering RNA (siRNA) stably expressed in A549 cells. The efficient reduction of caveolin-1 and -2 levels by siRNA has been confirmed by Western Blot. By using colorimetric assay and cell count, we are currently determining the possible differences in growth of A549 cell populations expressing control siRNA and caveolin-1 or -2 siRNA in the absence or presence of different concentrations of two anticancer drugs, i.e., taxol and etoposide. We hypothesize that in the absence of drugs, cells with a knockdown of caveolin-1 or -2 will grow equally as fast as or faster than control cells. Conversely, we expect that knockdown of caveolins will increase sensitivity of A549 to anti-cancer drugs and limit the possibility of developing drug resistance. Such results would be a first direct evidence that caveolin-1 and/or -2 are involved in acquiring drug resistance. If the latter is true, targeting caveolins in lung adenocarcinomas and other cancers could limit or even prevent the development of drug resistance and thus increase the efficacy of anti-cancer drugs

High-fat diet feeding alters metabolic response to fasting/non fasting conditions. Effect on caveolin expression and insulin signalling.

BACKGROUND: The effect of food intake on caveolin expression in relation to insulin signalling was studied in skeletal muscle and adipocytes from retroperitoneal (RP) and subcutaneous (SC) adipose tissue, comparing fasted (F) to not fasted (NF) rats that had been fed a control or high-fat (HF) diet for 72 days. METHODS: Serum glucose was analysed enzymatically and insulin and leptin by ELISA. Caveolins and insulin signalling intermediaries (IR, IRS-1 and 2 and GLUT4) were determined by RT-PCR and western blotting. Caveolin and IR phosphorylation was measured by immunoprecipitation. Data were analysed with Mann-Whitney U test. RESULTS: High-fat fed animals showed metabolic alterations and developed obesity and insulin resistance. In skeletal muscle, food intake (NF) induced activation of IR and increased expression of IRS-2 in control animals with normal metabolic response. HF animals became overweight, hyperglycaemic, hyperinsulinemic, hyperleptinemic and showed insulin resistance. In skeletal muscle of these animals, food intake (NF) also induced IRS-2 expression together with IR, although this was not active. Caveolin 3 expression in this tissue was increased by food intake (NF) in animals fed either diet. In RP adipocytes of control animals, food intake (NF) decreased IR and IRS-2 expression but increased that of GLUT4. A similar but less intense response was found in SC adipocytes. Food intake (NF) did not change caveolin expression in RP adipocytes with either diet, but in SC adipocytes of HF animals a reduction was observed. Food intake (NF) decreased caveolin-1 phosphorylation in RP but increased it in SC adipocytes of control animals, whereas it increased caveolin-2 phosphorylation in both types of adipocytes independently of the diet. CONCLUSIONS: Animals fed a control-diet show a normal response to food intake (NF), with activation of the insulin signalling pathway but without appreciable changes in caveolin expression, except a small increase of caveolin-3 in muscle. Animals fed a high-fat diet develop metabolic changes that result in insulin signalling impairment. In these animals, caveolin expression in muscle and adipocytes seems to be regulated independently of insulin signalling

The Biophysical Characterization of Caveolin-1

The main topic of this doctoral dissertation is the biophysical characterization of caveolin-1. Caveolin-1 is an integral membrane protein that has been shown to be essential for the formation of caveolae. Caveolae are 50-100 nm invaginations in the plasma membrane that have a plethora of cellular functions including signal transduction, relieving mechano-stresses on the cell, and endocytosis. Caveolin-1 is at the center of all of the functions of caveolae and has been shown to play a predominant role in disease states. However, while there are a large number of biological studies on caveolin-1, there are few biophysical studies, leading to a lack of understanding of the structure, topology and oligomerization of caveolin-1. The progress made in these three main areas of caveolin-1 research as well as introducing a novel in vitro functional assay for caveolin-1 and a broadly applicable membrane protein isolation technique are introduced. In chapter 1, background and general information about caveolin-1 and the biophysical techniques that were utilized for its characterization are discussed. Chapter 2 discusses the structural characterization of a caveolin-1 construct containing residues 62-136 using NMR spectroscopy revealing that the N-terminal residues (62-85) were dynamic and caveolin-1 contains a helix-break-helix motif with two approximately equal length helices. Chapter 3 discusses the structural characterization of caveolin-1 residues (62-178) using NMR spectroscopy. Caveolin-1(62-178) is the longest construct of caveolin-1 to be structurally characterized and encompasses the previously uncharacterized C-terminal domain which formed a long helix. Additionally, caveolin-1 contains a helix-break-helix-break-helix motif. In chapter 4, alanine and phenylalanine scanning mutagenesis of caveolin-1 82-136, was utilized to identify key structural residues within both helix-1 and helix-2. In chapter 5, the efforts to establish an in vitro functional assay for caveolin-1 utilizing the inhibition of endothelial nitric oxide synthase is presented. In chapter 6, cysteine scanning mutagenesis was utilized to evaluate the exposure of single residues in the caveolin-1 scaffolding domain to determine the topology of caveolin-1. Additionally, an evaluation of several different maleimide probes is presented. In chapter 7, a novel method to measure membrane protein oligomerization utilizing homo-FRET in liposomes is presented. Finally, in chapter 8 a purification method utilizing perfluorooctanoic acid (PFOA) to solubilize inclusion bodies is presented. This method has a three-fold advantage over conventional solubilization methods because: 1) PFOA can completely solubilize inclusion bodies, 2) PFOA is compatible with Ni-NTA chromatography and 3) PFOA is easily removed by detergent dialysis. Overall, this work represents significant advancements in understanding of the caveolin-1 protein