Characterization of the spontaneously transformed human endothelial cell line ECV304: 1. Multiple chromosomal rearrangements in ECV304 cells

Using differential G-staining of chromosomes, the karyotype of the endothelial cell line ECV304 obtained from endotheliocytes of the human umbilical vein was studied. The cells have been shown to have a polyploid karyotype with a number of chromosomes ranging from 96 to 112, as well as multiple numerical and structural clonal chromosome abnormalities. The structural rearrangements involve almost all chromosomes of the karyotype. Several paired chromosomal rearrangements have been revealed and include del(9)(p21), as well as two derivates of chromosome 3 with a breakpoint at the p25 locus, i.e., der(3)t(3;12)(3p25;12q11～12;12q21～24.?1) and der(3)t(3;?)(3p25). The role of these rearrangements in the immortalization of endotheliocytes and in angiogenesis is discussed. A comparison of the karyotypes of the cell line ECV304 and of the bladder carcinoma T24 cell line has shown that these karyotypes differ in all of the main cytogenetic characteristics. No identical structural chromosomal rearrangements, nor rearrangements characteristic of bladder carcinoma cells have been revealed. The studied endothelial cell line ECV304 is not identical to the T24 cell line.     

MAL2, a novel raft protein of the MAL family,is an essential component of the machinery for transcytosis in hepatoma HepG2 cells

Transcytosis is used alone (e.g., hepatoma HepG2 cells) or in combination with a direct pathway from the Golgi (e.g., epithelial MDCK cells) as an indirect route for targeting proteins to the apical surface. The raft-associated MAL protein is an essential element of the machinery for the direct route in MDCK cells. Herein, we present the functional characterization of MAL2, a member of the MAL protein family, in polarized HepG2 cells. MAL2 resided selectively in rafts and is predominantly distributed in a compartment localized beneath the subapical F-actin cytoskeleton. MAL2 greatly colocalized in subapical endosome structures with transcytosing molecules en route to the apical surface. Depletion of endogenous MAL2 drastically blocked transcytotic transport of exogenous polymeric immunoglobulin receptor and endogenous glycosylphosphatidylinositol-anchored protein CD59 to the apical membrane. MAL2 depletion did not affect the internalization of these molecules but produced their accumulation in perinuclear endosome elements that were accessible to transferrin. Normal transcytosis persisted in cells that expressed exogenous MAL2 designed to resist the depletion treatment. MAL2 is therefore essential for transcytosis in HepG2 cells.     

Characteristics of the spontaneously transformed human endothelial cell line ECV304. II. Functional responses of the ECV304 cells

Functional responses of the spontaneously transformed human endothelial cell line ECV304 were studied in order to asses its applicability as an endothelial cell model for studying angiogenesis and signal transduction. The dependence of proliferation activity of this line on the presence of growth factor was shown. The absent serum in culture medium resulted in blocking of cells in G1-phase of a cell cycle which is not typical for tumor cell lines. Low doses of beta particles emitted during [3H]thymidine decay resulted in blocking the proliferation of these cells in G2M-phase in a dose-dependent manner. Incubation of the cells with another source of beta particles, 3H2O, under condition of equal specific activities of tritium resulted in preferable accumulation of the cells in S-phase. The different efficiency of beta particles of tritium as a part of 3H2O molecule or thymidine demonstrates that various mechanisms are responsible for various check points. The check point of G1/S is absent and that complies with the presence of deletion of chromosome 9 in locus p21. The level of NO produced by constitutive form of NO-synthase in ECV304 cells was relatively low and not modified by inducible NO-synthase inhibitors. The data obtained suggest that ECV304 line cells retained the properties of the initial spontaneously transformed cell line obtained from human umbilical vein (HUVEC) as well as they can be used as a model system for further studies of the properties of vascular endothelial.     

Characteristics of the spontaneously transformed human endothelial cell line ECV304. I. Multiple chromosomal rearrangements in endothelial cells ECV304

Karyotype of endothelial line ECV304 cells obtained from human umbilicus vein endothelial cells was studied using G-banding chromosome staining. It has been revealed that the cells have a polyploidy karyotype with 96-112 chromosomes and multiple numerical and structural clonal rearrangements. Almost all the chromosomes of the karyotype are involved in structural rearrangements. There are several double chromosome rearrangements revealed including del(9)(p21) as well as two derivatives of chromosome 3 with the breakpoint in the locus p25 - der(3)t(3;12)(3p25;12q11- 12q24.?1) and der(3)t(3;?)(3p25). The role of these rearrangements in the immortalization of endothelial cells and sighs of transformation are discussed. In connection with the information received about the fact that the cells of ECV304 line are not endothelial cells but T24, urinary bladder cancer cells (which karyotype was studied by Hurst et al., 2000), the comparative analysis of the karyotypes of these two lines was carried out. It has been revealed that these two lines differ by all cytogenetic characteristics. Neither identical structural chromosomal rearrangements nor cell characteristic of urinary bladder cancer cells were detected. Our line ECV304 is not identical to the line T24.     

Identification and characterization of a novel gene EOLA1 stimulating ECV304 cell proliferation

To study the changes in gene expression in endothelial cells stimulated by lipopolysaccharide (LPS) we performed subtraction hybridization on control human umbilical vein endothelial cells (HUVEC) versus HUVEC stimulated by LPS. A novel cDNA, named endothelial-overexpressed lipopolysaccharide-associated factor 1 (EOLA1), was cloned from our differentially expressed EST database of HUVEC cDNA library (GenBank Accession No. ). Computational analysis showed that EOLA1 is 1404bp long, encoding a 158aa, 17.8kDa protein, mapped to chromosome Xq27.4 with 5 exons, expressed in different human normal tissues and cancer cell lines. Using the EOLA1 cDNA as bait, we performed a yeast two-hybrid screening of a human liver cDNA library and identified metallothionein 2A (MT2A) as associated protein. Stable transfection of EOLA1 stimulates ECV304 cell proliferation. Our data suggest that the physical interaction of EOLA1 and MT2A may have an important role of cell protection in inflammation reaction.     

Caveolin-1 interacts with a lipid raft-associated population of fatty acid synthase

Fatty Acid Synthase (FASN), a cytoplasmic biosynthetic enzyme, is the major source of long-chain fatty acids, particularly palmitate. Caveolin-1 (Cav-1) is a palmitoylated lipid raft protein that plays a key role in signal transduction and cholesterol transport. Both proteins have been implicated in prostate cancer (PCa) progression, and Cav-1 regulates FASN expression in a mouse model of aggressive PCa. We demonstrate that FASN and Cav-1 are coordinately up-regulated in human prostate tumors in a hormone-insensitive manner. Levels of FASN and Cav-1 protein expression discriminated between localized and metastatic cancers, and the two proteins exhibited analogous subcellular locations in a tumor subset. Endogenous FASN and Cav-1 were reciprocally co-immunoprecipitated from human and murine PCa cells, indicating that FASN forms a complex with Cav-1. FASN, a cytoplasmic enzyme, was induced to associate transiently with lipid raft membranes following alterations in signal transduction within the Src, Akt and EGFR pathways, suggesting that co-localization of FASN and Cav-1 is dependent on activation of upstream signaling mediators. A Cav-1 palmitoylation mutant, Cav-1C133/143/156S, that prevents phosphorylation by Src, did not interact with FASN. When overexpressed in Cav-1-negative PCa cells, Cav-1C133/143/156S caused a reduction of both Src and Akt levels, as well as of their active, phosphorylated forms, in comparison with wild type Cav-1. These findings suggest that FASN and Cav-1 physically and functionally interact in PCa cells. They also imply that palmitoylation within this complex is involved in tumor growth and survival.     

The cytoplasmic domain of influenza M2 protein interacts with caveolin-1

The cytoplasmic domain of influenza M2 protein (M2c) consists of 54 amino acid (aa) residues from aa44 to aa97. In this paper, M2c and its deletion mutant M2c_Δ47-55 were expressed using prokaryotic expression system. First, glutaraldehyde crosslinking assay showed that M2c had multimerization potential mediated by aa47-55. Then, M2c, instead of M2c_Δ47-55, directed eGFP from the whole cell localization to a predominately perinuclear region in CHO cells, which indicated that aa47-55 of M2c mediated the localization. Moreover, M2c colocalized with caveolin-1 (Cav) when CHO cells were cotransfected with Cav. A caveolin-1 binding motif ΦxxxxΦxxΦ (Φ represents aromatic amino acid residues) in aa47-55 of M2c was found by sequence alignment and analysis. Further overlay ELISA result showed that M2c, but not M2c_Δ47-55, bound to prokaryotically expressed cholesterol-free Cav_2-101, which illustrated the interaction could be cholesterol-independent. That was the first report of cellular protein bound to M2c.     

Vertebrate Arp6, a novel nuclear actin-related protein, interacts with heterochromatin protein 1

Actin-related proteins (Arps) were recently shown to contribute to the organization and regulation of chromatin structures. The nuclear functions of Arps have been investigated principally in budding yeast in which six of the ten Arp subfamilies are localized in the nucleus. In vertebrates, only two isoforms of Arp4 have so far been identified as showing localization to the nucleus. Here we show the predominant nuclear localization of another Arp subfamily, Arp6, in vertebrate cells. Vertebrate Arp6 directly interacted with heterochromatin protein 1 (HP1) orthologs and the two proteins colocalized in pericentric heterochromatin. Yeast Arp6 is involved in telomere silencing, while Drosophila Arp6 is localized in the pericentric heterochromatin. Our data strongly suggest that Arp6 has an evolutionarily conserved role in heterochromatin formation and also provide new insights into the molecular organization of heterochromatin.     

The rotavirus enterotoxin NSP4 directly interacts with the caveolar structural protein caveolin-1

Rotavirus nonstructural protein 4 (NSP4) is known to function as an intracellular receptor at the endoplasmic reticulum (ER) critical to viral morphogenesis and is the first characterized viral enterotoxin. Exogenously added NSP4 induces diarrhea in rodent pups and stimulates secretory chloride currents across intestinal segments as measured in Ussing chambers. Circular dichroism studies further reveal that intact NSP4 and the enterotoxic peptide (NSP4(114-135)) that is located within the extended, C-terminal amphipathic helix preferentially interact with caveola-like model membranes. We now show colocalization of NSP4 and caveolin-1 in NSP4-transfected and rotavirus-infected mammalian cells in reticular structures surrounding the nucleus (likely ER), in the cytosol, and at the cell periphery by laser scanning confocal microscopy. A direct interaction between NSP4 residues 112 to 140 and caveolin-1 was determined by the Pro-Quest yeast two-hybrid system with full-length NSP4 and seven overlapping deletion mutants as bait, caveolin-1 as prey, and vice versa. Coimmunoprecipitation of NSP4-caveolin-1 complexes from rotavirus-infected mammalian cells demonstrated that the interaction occurs during viral infection. Finally, binding of caveolin-1 from mammalian cell lysates to Sepharose-bound, NSP4-specific synthetic peptides confirmed the yeast two-hybrid data and further delineated the binding domain to amino acids 114 to 135. We propose that the association of NSP4 and caveolin-1 contributes to NSP4 intracellular trafficking from the ER to the cell surface and speculate that exogenously added NSP4 stimulates signaling molecules located in caveola microdomains.     

AFAP1L1 is a novel adaptor protein of the AFAP family that interacts with cortactin and localizes to invadosomes

The actin-filament associated protein (AFAP) family of adaptor proteins consists of three members: AFAP1, AFAP1L1, and AFAP1L2/XB130 with AFAP1 being the best described as a cSrc binding partner and actin cross-linking protein. A homology search of AFAP1 recently identified AFAP1L1 which has a similar sequence, domain structure and cellular localization; however, based upon sequence variations, AFAP1L1 is hypothesized to have unique functions that are distinct from AFAP1. While AFAP1 has the ability to bind to the SH3 domain of the nonreceptor tyrosine kinase cSrc via an N-terminal SH3 binding motif, it was unable to bind cortactin. However, the SH3 binding motif of AFAP1L1 was more efficient at interacting with the SH3 domain of cortactin and not cSrc. AFAP1L1 was shown by fluorescence microscopy to decorate actin filaments and move to punctate actin structures and colocalize with cortactin, consistent with localization to invadosomes. Upon overexpression in A7r5 cells, AFAP1L1 had the ability to induce podosome formation and move to podosomes without stimulation. Immunohistochemical analysis of AFAP1L1 in human tissues shows differential expression when contrasted with AFAP1 with localization of AFAP1L1 to unique sites in muscle and the dentate nucleus of the brain where AFAP1 was not detectable. We hypothesize AFAP1L1 may play a similar role to AFAP1 in affecting changes in actin filaments and bridging interactions with binding partners, but we hypothesize that AFAP1L1 may forge unique protein interactions in which AFAP1 is less efficient, and these interactions may allow AFAP1L1 to affect invadosome formation.     

H-Ras is a negative regulator of alpha3beta1 integrin expression in ECV304 endothelial cells

We have examined the role of Ras in integrin expression in ECV304 endothelial cells. Among the integrins examined in stable ECV304 transfectants expressing dominant active H-Ras (DAR-ECV), expression of alpha3beta1 integrin showed a prominent reduction in all the DAR-ECV clones when compared to the parental ECV304 cells. This implies that H-Ras negatively regulates the expression of alpha3beta1 integrin in ECV304 cells. When treated with inhibitors of the Ras downstream pathway (LY294002, PD98059, SB203580), the expression of alpha3beta1 integrin was up-regulated most significantly by LY294002, suggesting that among the downstream pathways of Ras, phosphatidylinositol 3-kinase is a major determinant. With the application of blocking antibody to alpha3beta1 integrin (2 - 2 x 10(4) nM), migration of ECV304 cells was enhanced to maximal (18%) at 20 nM. These results suggest that migration of endothelial cells could be modulated by H-Ras via alteration of the expression levels of alpha3beta1 integrin.     

The raft-associated protein MAL is required for maintenance of proper axon--glia interactions in the central nervous system

The myelin and lymphocyte protein (MAL) is a tetraspan raft-associated proteolipid predominantly expressed by oligodendrocytes and Schwann cells. We show that genetic ablation of mal resulted in cytoplasmic inclusions within compact myelin, paranodal loops that are everted away from the axon, and disorganized transverse bands at the paranode--axon interface in the adult central nervous system. These structural changes were accompanied by a marked reduction of contactin-associated protein/paranodin, neurofascin 155 (NF155), and the potassium channel Kv1.2, whereas nodal clusters of sodium channels were unaltered. Initial formation of paranodal regions appeared normal, but abnormalities became detectable when MAL started to be expressed. Biochemical analysis revealed reduced myelin-associated glycoprotein, myelin basic protein, and NF155 protein levels in myelin and myelin-derived rafts. Our results demonstrate a critical role for MAL in the maintenance of central nervous system paranodes, likely by controlling the trafficking and/or sorting of NF155 and other membrane components in oligodendrocytes.     

Integrin-linked kinase complexes with caveolin-1 in human neuroblastoma cells

Integrin-linked kinase (ILK) and caveolin-1 (cav-1) are implicated in the pathogenesis of cancer. Overexpression of ILK leads to altered expression of cell cycle regulators, a decreased level of cell adhesion to the extracellular matrix, a decreased level of apoptosis, in vitro phosphorylation of Akt, and tumor formation in nude mice. Conversely, cav-1 expression is frequently downregulated in many forms of cancer. We examined whether ILK and cav-1 interact in SHEP human neuroblastoma cells because ILK is present in caveolae-enriched membranes and contains a putative cav-binding domain. SHEP cells were stably transfected with vector, wild-type ILK (ILK-wt), kinase-deficient ILK (ILK-kd), or mutant cav-binding domain ILK (ILK-mutCavbd). Control SHEP cells and ILK transfectants express high levels of ILK and cav-1. Immunoprecipitation with anti-cav-1 co-immunoprecipitates a 59 kDa protein that is immunoreactive with the anti-ILK antibody, and this interaction is partially prevented in cells expressing ILK-mutCavbd. Cav-1 and ILK partially colocalize in SHEP cells, also supporting these data. Last, affinity chromatography with a biotinylated cav-scaffolding domain peptide precipitates ILK-wt but not ILK-mutCavbd. These data suggest that the cav-binding domain of ILK and the cav-scaffolding domain of cav-1 mediate complex formation in human neuroblastoma cells.     

Intelectin: a novel lipid raft-associated protein in the enterocyte brush border

Intelectin is a mammalian Ca2+-dependent, D-galactosyl-specific lectin expressed in Paneth and goblet cells of the small intestine and proposed to serve a protective role in the innate immune response to parasite infection. In addition, it is structurally identical to the intestinal lactoferrin receptor known to reside in the enterocyte brush border. To clarify this apparent discrepancy with regard to localization, the aim of this work was to study the cellular and subcellular distribution of small intestinal intelectin by immunofluorescence and immunogold electron microscopy. Secretory granules of lysozyme-positive Paneth cells in the bottom of the crypts as well as goblet cells along the crypt-villus axis were intensively labeled with intelectin antibodies, but quantitatively, the major site of intelectin deposition was the enterocyte brush border. This membrane is organized in stable glycolipid-based lipid raft microdomains, and like the divalent lectin galectin-4, intelectin was enriched in microvillar "superrafts", i.e., membranes that resist solubilization with Triton X-100 at 37 degrees C. This strategic localization suggests that the trimeric intelectin, like galectin-4, serves as an organizer and stabilizer of the brush border membrane, preventing loss of digestive enzymes to the gut lumen and protecting the glycolipid microdomains from pathogens.     

SLXL1, a novel acrosomal protein, interacts with DKKL1 and is involved in fertilization in mice

Background

Spermatogenesis is a complex cellular developmental process which involves diverse families of genes. The Xlr (X-linked, lymphocyte regulated) family includes multiple members, only a few of which have reported functions in meiosis, post-meiotic maturation, and fertilization of germ cells. Slx-like1 (Slxl1) is a member of the Xlr family, whose expression and function in spermatogenesis need to be elucidated.

Methodology/Principal Findings

The mRNA and protein expression and localization of Slxl1 were investigated by RT-PCR, Western blotting and immunohistochemistry in different tissues and at different stages of spermatogenesis. The interacting partner of SLXL1 was examined by co-immunoprecipitation and co-localization. Assessment of the role of SLXL1 in capacitation, acrosome reaction, zona pellucida binding/penetration, and fertilization was carried out in vitro using blocking antisera. The results showed that Slxl1 mRNA and protein were specifically expressed in the testis. SLXL1 was exclusively located in the acrosome of post-meiotic germ cells and interacts with DKKL1 (Dickkopf-like1), which is an acrosome-associated protein and plays an important role in fertilization. The rates of zona pellucida binding/penetration and fertilization were significantly reduced by the anti-SLXL1 polyclonal antiserum.

Conclusions/Significance

SLXL1 is the first identified member of the XLR family that is associated with acrosome and is involved in zona pellucid binding/penetration and subsequent fertilization. These results, together with previous studies, suggest that Xlr family members participate in diverse processes from meiosis to fertilization during spermatogenesis.     

Identification of PLSCR1 as a protein that interacts with RELT family members

Receptor expressed in lymphoid tissues (RELT) proteins are recently described surface receptors belonging to the larger TNF receptor family. To improve our understanding of RELT-mediated signal transduction, we performed a screen for RELT-interacting proteins. Phospholipid Scramblase 1 (PLSCR1) was identified through a yeast two-hybrid genetic screen utilizing the intracellular portion of the RELT family member, RELL1, as bait. PLSCR1 was observed to physically interact with all known RELT family members as determined by co-immunoprecipitation experiments. The protein kinase, oxidative stress responsive 1 (OSR1) was previously shown to interact and phosphorylate all three RELT family members. In our study, no physical association was observed between OSR1 and PLSCR1 alone. However, in the presence of RELT, OSR1 was capable of co-immunoprecipitating PLSCR1, suggesting the formation of a protein complex between RELT, OSR1, and PLSCR1. In addition, OSR1 phosphorylated PLSCR1 in an in vitro kinase assay, but only in the presence of RELT, suggesting a functional multiprotein complex. RELT and PLSCR1 co-localized in intracellular regions of human embryonic kidney-293 cells, with RELT overexpression appearing to alter the localization of PLSCR1. These studies demonstrate that RELT family members physically interact with PLSCR1, and that these interactions may regulate the phosphorylation of PLSCR1 by OSR1.