Caveolar Fatty Acids and Acylation of Caveolin-1

Purpose

Caveolae are cholesterol and sphingolipids rich subcellular domains on plasma membrane. Caveolae contain a variety of signaling proteins which provide platforms for signaling transduction. In addition to enriched with cholesterol and sphingolipids, caveolae also contain a variety of fatty acids. It has been well-established that acylation of protein plays a pivotal role in subcellular location including targeting to caveolae. However, the fatty acid compositions of caveolae and the type of acylation of caveolar proteins remain largely unknown. In this study, we investigated the fatty acids in caveolae and caveolin-1 bound fatty acids.

Methods

Caveolae were isolated from Chinese hamster ovary (CHO) cells. The caveolar fatty acids were extracted with Folch reagent, methyl esterificated with BF3, and analyzed by gas chromatograph-mass spectrometer (GC/MS). The caveolin-1bound fatty acids were immunoprecipitated by anti-caveolin-1 IgG and analyzed with GC/MS.

Results

In contrast to the whole CHO cell lysate which contained a variety of fatty acids, caveolae mainly contained three types of fatty acids, 0.48 µg palmitic acid, 0.61 µg stearic acid and 0.83 µg oleic acid/caveolae preparation/5×10⁷ cells. Unexpectedly, GC/MS analysis indicated that caveolin-1 was not acylated by myristic acid; instead, it was acylated by palmitic acid and stearic acid.

Conclusion

Caveolae contained a special set of fatty acids, highly enriched with saturated fatty acids, and caveolin-1 was acylated by palmitic acid and stearic acid. The unique fatty acid compositions of caveolae and acylation of caveolin-1 may be important for caveolae formation and for maintaining the function of caveolae.     

Dkk-1 Inhibits Intestinal Epithelial Cell Migration by Attenuating Directional Polarization of Leading Edge Cells

Wnt signaling pathways regulate proliferation, motility, and survival in a variety of human cell types. Dickkopf-1 (Dkk-1) is a secreted Wnt antagonist that has been proposed to regulate tissue homeostasis in the intestine. In this report, we show that Dkk-1 is secreted by intestinal epithelial cells after wounding and that it inhibits cell migration by attenuating the directional orientation of migrating epithelial cells. Dkk-1 exposure induced mislocalized activation of Cdc42 in migrating cells, which coincided with a displacement of the polarity protein Par6 from the leading edge. Consequently, the relocation of the microtubule organizing center and the Golgi apparatus in the direction of migration was significantly and persistently inhibited in the presence of Dkk-1. Small interfering RNA-induced down-regulation of Dkk-1 confirmed that extracellular exposure to Dkk-1 was required for this effect. Together, these data demonstrate a novel role of Dkk-1 in the regulation of directional polarization of migrating intestinal epithelial cells, which contributes to the effect of Dkk-1 on wound closure in vivo.     

Modulation of Dendritic Cell Activation and Subsequent Th1 Cell Polarization by Lidocaine

Dendritic cells play an essential role in bridging innate and adaptive immunity by recognizing cellular stress including pathogen- and damage-associated molecular patterns and by shaping the types of antigen-specific T cell immunity. Although lidocaine is widely used in clinical settings that trigger cellular stress, it remains unclear whether such treatment impacts the activation of innate immune cells and subsequent differentiation of T cells. Here we showed that lidocaine inhibited the production of IL–6, TNFα and IL–12 from dendritic cells in response to toll-like receptor ligands including lipopolysaccharide, poly(I:C) and R837 in a dose-dependent manner. Notably, the differentiation of Th1 cells was significantly suppressed by the addition of lidocaine while the same treatment had little effect on the differentiation of Th17, Th2 and regulatory T cells in vitro. Moreover, lidocaine suppressed the ovalbumin-specific Th1 cell responses in vivo induced by the adoptive transfer of ovalbumin-pulsed dendritic cells. These results demonstrate that lidocaine inhibits the activation of dendritic cells in response to toll-like receptor signals and subsequently suppresses the differentiation of Th1 cell responses.     

Nox4 and Duox1/2 Mediate Redox Activation of Mesenchymal Cell Migration by PDGF

Platelet derived growth factor (PDGF) orchestrates wound healing and tissue regeneration by regulating recruitment of the precursor mesenchymal stromal cells (MSC) and fibroblasts. PDGF stimulates generation of hydrogen peroxide that is required for cell migration, but the sources and intracellular targets of H₂O₂ remain obscure. Here we demonstrate sustained live responses of H₂O₂ to PDGF and identify PKB/Akt, but not Erk1/2, as the target for redox regulation in cultured 3T3 fibroblasts and MSC. Apocynin, cell-permeable catalase and LY294002 inhibited PDGF-induced migration and mitotic activity of these cells indicating involvement of PI3-kinase pathway and H₂O₂. Real-time PCR revealed Nox4 and Duox1/2 as the potential sources of H₂O₂. Silencing of Duox1/2 in fibroblasts or Nox4 in MSC reduced PDGF-stimulated intracellular H₂O₂, PKB/Akt phosphorylation and migration, but had no such effect on Erk1/2. In contrast to PDGF, EGF failed to increase cytoplasmic H₂O₂, phosphorylation of PKB/Akt and migration of fibroblasts and MSC, confirming the critical impact of redox signaling. We conclude that PDGF-induced migration of mesenchymal cells requires Nox4 and Duox1/2 enzymes, which mediate redox-sensitive activation of PI3-kinase pathway and PKB/Akt.     

Calponin 2 Acts As an Effector of Noncanonical Wnt-Mediated Cell Polarization during Neural Crest Cell Migration

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Highlights? Cnn2 is expressed in NCCs and required for their migration in frogs and chicks ? Cnn2 is inactivated by noncanonical Wnt signaling ? Loss of Cnn2 causes a switch from cortical actin to central stress fibers ? Cnn2 polarizes the actin cytoskeleton downstream of PCP     

Role for Phospholipid Flippase Complex of ATP8A1 and CDC50A Proteins in Cell Migration

Type IV P-type ATPases (P4-ATPases) and CDC50 family proteins form a putative phospholipid flippase complex that mediates the translocation of aminophospholipids such as phosphatidylserine (PS) and phosphatidylethanolamine (PE) from the outer to inner leaflets of the plasma membrane. In Chinese hamster ovary (CHO) cells, at least eight members of P4-ATPases were identified, but only a single CDC50 family protein, CDC50A, was expressed. We demonstrated that CDC50A associated with and recruited P4-ATPase ATP8A1 to the plasma membrane. Overexpression of CDC50A induced extensive cell spreading and greatly enhanced cell migration. Depletion of either CDC50A or ATP8A1 caused a severe defect in the formation of membrane ruffles, thereby inhibiting cell migration. Analyses of phospholipid translocation at the plasma membrane revealed that the depletion of CDC50A inhibited the inward translocation of both PS and PE, whereas the depletion of ATP8A1 inhibited the translocation of PE but not that of PS, suggesting that the inward translocation of cell-surface PE is involved in cell migration. This hypothesis was further examined by using a PE-binding peptide and a mutant cell line with defective PE synthesis; either cell-surface immobilization of PE by the PE-binding peptide or reduction in the cell-surface content of PE inhibited the formation of membrane ruffles, causing a severe defect in cell migration. These results indicate that the phospholipid flippase complex of ATP8A1 and CDC50A plays a major role in cell migration and suggest that the flippase-mediated translocation of PE at the plasma membrane is involved in the formation of membrane ruffles to promote cell migration.     

A Modeling Approach to Study the Effect of Cell Polarization on Keratinocyte Migration

The skin forms an efficient barrier against the environment, and rapid cutaneous wound healing after injury is therefore essential. Healing of the uppermost layer of the skin, the epidermis, involves collective migration of keratinocytes, which requires coordinated polarization of the cells. To study this process, we developed a model that allows analysis of live-cell images of migrating keratinocytes in culture based on a small number of parameters, including the radius of the cells, their mass and their polarization. This computational approach allowed the analysis of cell migration at the front of the wound and a reliable identification and quantification of the impaired polarization and migration of keratinocytes from mice lacking fibroblast growth factors 1 and 2 – an established model of impaired healing. Therefore, our modeling approach is suitable for large-scale analysis of migration phenotypes of cells with specific genetic defects or upon treatment with different pharmacological agents.     

Genetic Variants in Caveolin-1 and RhoA/ROCK1 Are Associated with Clear Cell Renal Cell Carcinoma Risk in a Chinese Population

Background

The RhoA/ROCK pathway and Caveolin-1 (Cav-1) participate in the process of tumorigenesis in numerous types of cancer. Up-regulation of RhoA/ROCK and Cav-1 expression is considered to be associated with the development and progression of clear cell renal cell carcinoma (ccRCC). We investigated the association between genetic variations of RhoA/ROCK and Cav-1 and the risk of ccRCC in the Chinese population.

Methods

Between May 2004 and March 2014, a total of 1,248 clear cell renal cell carcinoma cases and 1,440 cancer-free controls were enrolled in this hospital-based case-control study. Nine SNPs in RhoA/ROCK and Cav-1 were genotyped using the TaqMan assay.

Result

We found two SNPs (Cav-1 rs1049334 and ROCK1 rs35996865) were significantly associated with the increasing risk of ccRCC (P = 0.002 and P < 0.001 respectively). The analysis of combined risk alleles revealed that patients with 2–4 risk alleles showed a more remarkable growth of ccRCC risk than the patients with 0–1 risk alleles(OR = 1.66, 95%CI = 1.31–2.11, P < 0.001). Younger subjects (P = 0.001, OR = 1.83, 95%CI = 1.30–2.57), higher weight subjects (P = 0.001, OR = 1.76, 95%CI = 1.25–2.47), female subjects (P = 0.007, OR = 1.75, 95% CI = 1.17–2.62), nonsmokers (P < 0.001, OR = 1.67, 95%CI = 1.26–2.23), drinkers (P = 0.025, OR = 1.75, 95% CI = 1.07–2.85), subjects with hypertension (P = 0.025, OR = 1.75, 95% CI = 1.07–2.85) and diabetes (P = 0.026, OR = 4.31, 95% CI = 1.19–15.62) showed a stronger association between the combined risk alleles and the risk of ccRCC by using the stratification analysis. Furthermore, we observed higher Cav-1 mRNA levels in the presence of the rs1049334 A allele in normal renal tissues.

Conclusion

Our results indicate that the two SNPs (Cav-1 rs1049334 and ROCK1 rs35996865) and genotypes with a combination of 2–4 risk alleles were associated with the risk of ccRCC. The functional SNP rs1049334 may affect the risk of ccRCC by altering the expression of Cav-1 and the relevance between the risk effects and the functional impact of this polymorphism needs further validation.     

Inhibition of Caveolin-1 Restores Myeloid Cell Function in Human Glioblastoma

Background

Gliomas are the most common primary brain tumor in both children and adults. The prognosis for glioblastoma (GBM), the most common type of malignant glioma, has remained dismal, with median survival a little over one year despite maximal therapy with surgery, chemotherapy, and radiation. Although immunotherapy has become increasingly successful against many systemic tumors, clinical efficacy against brain tumors has been limited. One reason for this is an incomplete understanding of the local immunologic tumor microenvironment, particularly the function of large numbers of infiltrating myeloid derived cells. Monocytes/microglia are myeloid derived immunomodulatory cells, and they represent the predominant infiltrating immune cell population in gliomas. Our group has previously demonstrated using complementary in vitro and in vivo approaches that GBM tumor cells polarize tumor-associated myeloid cells (TAMs) and suppress their immunostimulatory function.

Methods and Results

To better understand the mechanisms responsible for this immunosuppression, we used gene expression profiling of stimulated monocytes in the presence or absence of GBM tumor cells. Our analysis identified caveolin-1 (CAV1), a plasma membrane molecule with pleiotropic functions, as significantly up-regulated in monocytes in the presence of GBMs. We validated these findings ex vivo by confirming up-regulation of CAV1 in TAMs isolated from GBMs immediately after surgical resection. Finally, we demonstrate that siRNA inhibition of CAV1 restores myeloid cell function, as measured by TNF-alpha secretion, in the presence of GBMs.

Conclusions

Restoration of TAM function through pharmacologic blockage of CAV1 may facilitate more successful immunotherapeutic strategies directed against a variety of solid human tumors infiltrated by TAMs.     

Neuronal Differentiation Dictates Estrogen-Dependent Survival and ERK1/2 Kinetic by Means of Caveolin-1

Estrogens promote a plethora of effects in the CNS that profoundly affect both its development and mature functions and are able to influence proliferation, differentiation, survival and neurotransmission. The biological effects of estrogens are cell-context specific and also depend on differentiation and/or proliferation status in a given cell type. Furthermore, estrogens activate ERK1/2 in a variety of cellular types. Here, we investigated whether ERK1/2 activation might be influenced by estrogens stimulation according to the differentiation status and the molecular mechanisms underling this phenomenon. ERK1/2 exert an opposing role on survival and death, as well as on proliferation and differentiation depending on different kinetics of phosphorylation. Hence we report that mesencephalic primary cultures and the immortalized cell line mes-c-myc A1 express estrogen receptor α and activate ERK1/2 upon E₂ stimulation. Interestingly, following the arrest of proliferation and the onset of differentiation, we observe a change in the kinetic of ERKs phosphorylation induced by estrogens stimulation. Moreover, caveolin-1, a main constituent of caveolae, endogenously expressed and co-localized with ER-α on plasma membrane, is consistently up-regulated following differentiation and cell growth arrest. In addition, we demonstrate that siRNA-induced caveolin-1 down-regulation or disruption by means of ß-cyclodextrin treatment changes ERK1/2 phosphorylation in response to estrogens stimulation. Finally, caveolin-1 down-regulation abolishes estrogens-dependent survival of neurons. Thus, caveolin-1 appears to be an important player in mediating, at least, some of the non-genomic action of estrogens in neurons, in particular ERK1/2 kinetics of activation and survival.     

Golgi Anti-apoptotic Proteins Are Highly Conserved Ion Channels That Affect Apoptosis and Cell Migration

Golgi anti-apoptotic proteins (GAAPs) are multitransmembrane proteins that are expressed in the Golgi apparatus and are able to homo-oligomerize. They are highly conserved throughout eukaryotes and are present in some prokaryotes and orthopoxviruses. Within eukaryotes, GAAPs regulate the Ca²⁺ content of intracellular stores, inhibit apoptosis, and promote cell adhesion and migration. Data presented here demonstrate that purified viral GAAPs (vGAAPs) and human Bax inhibitor 1 form ion channels and that vGAAP from camelpox virus is selective for cations. Mutagenesis of vGAAP, including some residues conserved in the recently solved structure of a related bacterial protein, BsYetJ, altered the conductance (E207Q and D219N) and ion selectivity (E207Q) of the channel. Mutation of residue Glu-207 or -178 reduced the effects of GAAP on cell migration and adhesion without affecting protection from apoptosis. In contrast, mutation of Asp-219 abrogated the anti-apoptotic activity of GAAP but not its effects on cell migration and adhesion. These results demonstrate that GAAPs are ion channels and define residues that contribute to the ion-conducting pore and affect apoptosis, cell adhesion, and migration independently.     

Choline Plasmalogens Isolated from Swine Liver Inhibit Hepatoma Cell Proliferation Associated with Caveolin-1/Akt Signaling

Plasmalogens play multiple roles in the structures of biological membranes, cell membrane lipid homeostasis and human diseases. We report the isolation and identification of choline plasmalogens (ChoPlas) from swine liver by high performance thin layer chromatography (HPTLC) and high performance liquid chromatography (HPLC)/MS. The growth and viability of hepatoma cells (CBRH7919, HepG2 and SMMC7721) was determined following ChoPlas treatment comparing with that of human normal immortal cell lines (HL7702). Result indicated that ChoPlas inhibited hepatoma cell proliferation with an optimal concentration and time of 25 μmol/L and 24 h. To better understand the mechanism of the ChoPlas-induced inhibition of hepatoma cell proliferation, Caveolin-1 and PI3K/Akt pathway signals, including total Akt, phospho-Akt(pAkt) and Bcl-2 expression in CBRH7919 cells, were determined by western blot. ChoPlas treatment increased Caveolin-1 expression and reduced the expression of phospho-Akt (pAkt) and Bcl-2, downstream targets of the PI3K/Akt pathway. Further cell cycle analysis showed that ChoPlas treatment induced G₁ and G₁/S phase transition cell cycle arrest. The expression of essential cell cycle regulatory proteins involved in the G₁ and G₁/S phase transitions, cyclin D, CDK4, cyclin E and CDK2, were also analyzed by western blot. ChoPlas reduced CDK4, cyclin E and CDK2 expression. Taken together, the results indicate that swine liver-derived natural ChoPlas inhibits hepatoma cell proliferation associated with Caveolin-1 and PI3K/Akt signals.