Knockdown of flotillin-2 inhibits lung surfactant secretion by alveolar type II cells

Chintagari, Narendranath Reddy; Gou, Deming; Liu, Lin

doi:10.1038/cr.2008.55

Cited by 6 publications

(8 citation statements)

References 10 publications

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“…1A and B). This finding is consistent with previous report [33] and is likely to reflects known hetero- oligomerization of flotillin-1 and flotillin-2 which stabilizes them at cell membranes [16,12]. More interestingly, we observed that downregulation of flotillin-1 induced a substantial (~65%) decrease in the level of caveolin-1, whereas flotillin-2 knock-down did not affect caveolin-1 expression (Fig.…”

Section: Resultssupporting

confidence: 93%

Flotillin-1 stabilizes caveolin-1 in intestinal epithelial cells

Vassilieva

Ivanov

Nusrat

2009

Biochemical and Biophysical Research Communications

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Flotillins and caveolins represent two types of resident proteins associated with lipid rafts in mammalian cells, however, their possible cross-talk in regulating lipid raft functions remains poorly understood. In this report, we observed that siRNA-mediated down-regulation of flotillin-1 expression which disrupted lipid raft-mediated endocytosis of BODIPY FL C 5 -lactosylceramide also substantially decreased caveolin-1 level in SK-CO15 human intestinal epithelial cells. The decrease in caveolin-1 expression appeared to be specific for flotillin-1 knock-down and was not observed after down-regulation of flotillin-2. The decrease in caveolin-1 level in flotillin-1-depleted cells was not due to suppression of its mRNA synthesis and was not mimicked by cholesterol depletion of SK-CO15 cells. Furthermore, flotillin-1 dependent down-regulation of caveolin-1 was reversed after cell exposure to lysosomal inhibitor, chloroquine but not proteosomal inhibitor, MG262. Our data suggest that flotillin-1 regulates caveolin-1 level by preventing its lysosomal degradation in intestinal epithelial cells. Keywords Epithelial cells; Lipid rafts; Lactosyl ceramide internalization; Lysosomal degradationThe lipid rafts represent specialized dynamic areas (domains) of eukaryotic membranes which are characterized by unique chemical composition and physical properties [1,2]. The most vivid feature of lipid raft is their enrichment in particular lipids such as cholesterol and sphingolipids, which create a microenvironment for recruitment of various membrane and cytosolic proteins with high binding affinity to these lipids. By clustering/segregating different lipid and protein constituents of the membrane, rafts mediate many crucial cellular functions, including regulation of signaling by plasma membrane receptors, activity of ion pumps and channels and mediation of vesicle trafficking/fusion [3,4]. Although membrane rafts are readily formed in a pure lipid environments of liposomes, in living cells these lipid domains are frequently supported by specialized proteins which are important modulators of raft structure, stability and functions [5]. Resident lipid raft proteins flotillins and caveolins are co-expressed in many types of mammalian cells, and as such they are frequently enriched in the same cell membranes. However, several recent studies have demonstrated that flotillins and caveolins do not intermix and populate distinct subsets of lipid rafts [6][7][8][9][10].The flotillin (also known as the reggie) protein family consists of two ubiquitously expressed isoforms, flotillin-1 and flotillin-2. Flotillins are evolutionary highly conserved proteins [11] *Corresponding author. Fax: +1 404 727 3321. anusrat@emory.edu (A. Nusrat). [12,13]. Flotillins are enriched at the plasma membrane where they associate with the inner leaflet via hydrophobic amino acid stretches and posttranslationally attached acyl groups of palmitic and myristic acids [13][14][15]. At the plasma membrane, flotillin-1 and flotillin-2 are organized as homo-...

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Section: Resultssupporting

confidence: 93%

Flotillin-1 stabilizes caveolin-1 in intestinal epithelial cells

Vassilieva

Ivanov

Nusrat

2009

Biochemical and Biophysical Research Communications

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“…Immunoblots of total protein extracted from cells transfected with siRNAs against human FLOT1 showed greater than 90% loss of flotillin-1 and approximately 80% loss of flotillin-2 protein, similar to previous reports (40)(41)(42) (Figure 5A; also see Supplemental Figure 5D). The mechanism by which suppression of either flotillin-1 or flotillin-2 leads to loss of the other is not understood.…”

Section: Figuresupporting

confidence: 66%

“…This result is likely explained by the finding that suppression of either reggie/flotillin leads to loss of the other (Supplemental Figure 5D; also shown in mammalian cells below and in refs. [40][41][42]. Together, our data show that the reggies/flotillins play a substantial role in CT action in vivo in zebrafish embryos.…”

Section: Figurementioning

confidence: 55%

Intoxication of zebrafish and mammalian cells by cholera toxin depends on the flotillin/reggie proteins but not Derlin-1 or -2

Saslowsky¹,

Cho²,

Chinnapen³

et al. 2010

J. Clin. Invest.

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Cholera toxin (CT) causes the massive secretory diarrhea associated with epidemic cholera. To induce disease, CT enters the cytosol of host cells by co-opting a lipid-based sorting pathway from the plasma membrane, through the trans-Golgi network (TGN), and into the endoplasmic reticulum (ER). In the ER, a portion of the toxin is unfolded and retro-translocated to the cytosol. Here, we established zebrafish as a genetic model of intoxication and examined the Derlin and flotillin proteins, which are thought to be usurped by CT for retro-translocation and lipid sorting, respectively. Using antisense morpholino oligomers and siRNA, we found that depletion of Derlin-1, a component of the Hrd-1 retro-translocation complex, was dispensable for CT-induced toxicity. In contrast, the lipid raft-associated proteins flotillin-1 and -2 were required. We found that in mammalian cells, CT intoxication was dependent on the flotillins for trafficking between plasma membrane/endosomes and two pathways into the ER, only one of which appears to intersect the TGN. These results revise current models for CT intoxication and implicate protein scaffolding of lipid rafts in the endosomal sorting of the toxin-GM1 complex. IntroductionCholera toxin (CT) is an AB 5 -subunit toxin responsible for the massive secretory diarrhea seen in epidemic cholera. As for most toxins, CT must gain access to the cytosol of host cells to cause disease. The strategy employed by CT is to bind ganglioside GM1 in the plasma membrane (PM) via the B-subunit (CTB). GM1 carries the toxin retrograde through endosomes, the trans-Golgi network (TGN), and likely all the way into the ER (1, 2). In the ER, a portion of the A-subunit (CTA), the A1-chain, crosses to the cytosol by coopting the machinery that retro-translocates terminally misfolded proteins for degradation by the proteasome (termed ER-associated degradation [ERAD]; refs. 3, 4). The A1-chain refolds in the cytosol and activates adenylate cyclase to increase cAMP. The mechanisms for lipid sorting and ERAD usurped by CT are fundamental to eukaryotic cell biology but remain incompletely understood. To explore how CT exploits these pathways in an unbiased way, we used the zebrafish as a model because it is amenable to genetic screens. Here, we show that CT intoxicates zebrafish embryos by hijacking the same basic mechanisms used in mammalian cells and examine the dependence of CT toxicity on two families of proteins implicated in toxin action: the flotillins and Derlins. These proteins have emerged as important components of lipid-based trafficking and ERAD, respectively.There is evidence that GM1 sorts CT retrograde from PM to ER by association with lipid rafts (2, 5-8). Lipid rafts are cooperative selfassemblies of lipids and proteins that influence various aspects of

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“…Stimulation of surfactant secretion by some agents increase membrane association of annexin VII and its colocalization with ABCA3 [217]. Further, the lipid raft marker, flotillin-2, regulates surfactant secretion via cytoskeletal rearrangement as flotillin-2 depletion in cells blocks the G-protein coupled receptor-mediated activation of p38 mitogen-activated protein kinases [218]. Besides these stimulatory factors, surfactant phospholipid secretion can be regulated by inhibitory factors.…”

Section: Surfactant Secretion and Regulationmentioning

confidence: 99%

Surfactant phospholipid metabolism

Agassandian

Mallampalli

2013

Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biolog

204

176

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Pulmonary surfactant is essential for life and is comprised of a complex lipoprotein-like mixture that lines the inner surface of the lung to prevent alveolar collapse at the end of expiration. The molecular composition of surfactant depends on highly integrated and regulated processes involving its biosynthesis, remodeling, degradation, and intracellular trafficking. Despite its multicomponent composition, the study of surfactant phospholipid metabolism has focused on two predominant components, disaturated phosphatidylcholine that confers surface-tension lowering activities, and phosphatidylglycerol, recently implicated in innate immune defense. Future studies providing a better understanding of the molecular control and physiological relevance of minor surfactant lipid components are needed.

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Knockdown of flotillin-2 inhibits lung surfactant secretion by alveolar type II cells

Cited by 6 publications

References 10 publications

Flotillin-1 stabilizes caveolin-1 in intestinal epithelial cells

Flotillin-1 stabilizes caveolin-1 in intestinal epithelial cells

Intoxication of zebrafish and mammalian cells by cholera toxin depends on the flotillin/reggie proteins but not Derlin-1 or -2

Surfactant phospholipid metabolism

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