ß2 spectrin-mediated differentiation repressed the properties of liver cancer stem cells through ß-catenin.

ßII-Spectrin (ß2SP), a Smad3/4 adaptor protein during transforming growth factor (TGF) ß/Smad signal pathway, plays a critical role in suppressing hepatocarcinogenesis. Dedifferentiation is a distinctive feature of cancer progression. Therefore, we investigated whether the disruption of ß2SP contributed to tumorigenesis of hepatocellular carcinoma (HCC) through the dedifferentiation. Down-regulation of ß2SP in hepatocytes was observed in cirrhotic liver and HCC. The level of ß2SP expression was closely associated with the differentiation status of hepatocytes in rat model of hepatocarcinogenesis and clinical specimens. Transgenic expression of ß2SP in HCC cells promoted the differentiation of HCC cells and suppressed the growth of HCC cells in vitro. Efficient transduction of ß2SP into liver CSCs resulted in a reduction in colony formation ability, spheroid formation capacity, invasive activity, chemo-resistance properties, tumorigenicity in vivo. In addition, ß2 spectrin exerted its effect through ß catenin in liver CSCs. In conclusion, ß2 spectrin repressed the properties of liver CSCs through inducing differentiation; thus, strategies to restore its levels and activities would be a novel strategy for HCC prevention and differentiation therapy.

Genetic studies of spectrin in the larval fat body of Drosophila melanogaster: evidence for a novel lipid uptake apparatus.

Spectrin cytoskeleton defects produce a host of phenotypes affecting the plasma membrane, cell polarity, and secretory membrane traffic. However, many of the underlying molecular mechanisms remain unexplained by prevailing models. Here we used the larval fat body of Drosophila melanogaster as a genetic model system to further elucidate mechanisms of αß-spectrin function. The results provide unexpected new insights into spectrin function as well as mechanisms of dietary fat uptake and storage. We show that loss of α- or ß-spectrin in the fat body eliminated a population of small cortical lipid droplets and altered plasma membrane architecture, but did not affect viability of the organism. We present a novel model in which αß-spectrin directly couples lipid uptake at the plasma membrane to lipid droplet growth in the cytoplasm. In contrast, strong overexpression of ß-spectrin caused fat body atrophy and larval lethality. Overexpression of ß-spectrin also perturbed transport of dietary fat from the midgut to the fat body. This hypermorphic phenotype appears to be the result of blocking secretion of the lipid carrier lipophorin from fat cells. However, this midgut phenotype was never seen with spectrin loss of function, suggesting that spectrin is not normally required for lipophorin secretion or function. The ß-spectrin hypermorphic phenotype was ameliorated by co-overexpression of α-spectrin. Based on the overexpression results here, we propose that ß-spectrin family members may be prone to hypermorphic effects (including effects on secretion) if their activity is not properly regulated.

Non-erythroid alpha spectrin prevents telomere dysfunction after DNA interstrand cross-link damage.

Telomere integrity is critical for telomere function and genomic stability. We previously demonstrated that non-erythroid α-spectrin (αIISp) is present in mammalian cell nuclei where it is important in repair of DNA interstrand cross-links (ICLs) and chromosome stability. We now demonstrate that αIISp is also important for telomere maintenance after ICL damage. It localizes to telomeres in S phase after ICL damage where it has enhanced association with TRF1 and TRF2 and is required for recruitment of the ICL repair protein, XPF, to damage-induced foci at telomeres. In telomerase-positive normal cells depleted of αIISp by siRNA or in Fanconi anemia, complementation group A (FA-A) cells, where αIISp levels are 35-40% of normal, ICL damage results in failure of XPF to localize to telomeres, markedly increased telomere dysfunction-induced foci, followed by catastrophic loss of telomeres. Restoration of αIISp levels to normal in FA-A cells corrects these deficiencies. Our studies demonstrate that αIISp is critical for repair of DNA ICLs at telomeres, likely by facilitating the recruitment of repair proteins similar, but not identical, to its proposed role in repair of DNA ICLs in genomic DNA and that this function in turn is critical for telomere maintenance after DNA ICL damage.

IL-27 inhibits HIV-1 infection in human macrophages by down-regulating host factor SPTBN1 during monocyte to macrophage differentiation.

The susceptibility of macrophages to HIV-1 infection is modulated during monocyte differentiation. IL-27 is an anti-HIV cytokine that also modulates monocyte activation. In this study, we present new evidence that IL-27 promotes monocyte differentiation into macrophages that are nonpermissive for HIV-1 infection. Although IL-27 treatment does not affect expression of macrophage differentiation markers or macrophage biological functions, it confers HIV resistance by down-regulating spectrin ß nonerythrocyte 1 (SPTBN1), a required host factor for HIV-1 infection. IL-27 down-regulates SPTBN1 through a TAK-1-mediated MAPK signaling pathway. Knockdown of SPTBN1 strongly inhibits HIV-1 infection of macrophages; conversely, overexpression of SPTBN1 markedly increases HIV susceptibility of IL-27-treated macrophages. Moreover, we demonstrate that SPTBN1 associates with HIV-1 gag proteins. Collectively, our results underscore the ability of IL-27 to protect macrophages from HIV-1 infection by down-regulating SPTBN1, thus indicating that SPTBN1 is an important host target to reduce HIV-1 replication in one major element of the viral reservoir.

Spectrin αII and ßII tetramers contribute to platinum anticancer drug resistance in ovarian serous adenocarcinoma.

To overcome drug resistance in ovarian carcinoma, novel resistance mechanisms must be elucidated for clinical application. We purified 2 proteins in the 300 kDa range from cisplatin-resistant cells (NOS2CR2) by affinity chromatography with cisplatin-exposed Glutathione Sepharose 4B. The purified proteins were identified as spectrin αII and ßII by peptide mass mapping analysis. Western blot analysis detected greater expression of spectrin αII and ßII in NOS2CR2 than in wild-type cells (NOS2). The same result was obtained for spectrin ßII expression by immunohistochemical staining. To determine whether spectrin αII and ßII contribute to resistance, a drug sensitivity test was performed on SKOV3 ovarian cancer cells transfected with small interfering RNA. Sensitivity to platinum drugs was increased in the expression reduced cells. In a clinical study of five ovarian serous adenocarcinoma cases, tumor specimens taken after treatment with carboplatin stained more strongly for spectrin ßII expression than untreated specimens. Fifty-two tumor specimens from 46 patients with ovarian serous adenocarcinoma were immunohistochemically stained for spectrin ßII and scored. Tumors previously treated by chemotherapy scored higher than those not treated. Of 27 cases with detectable residual tumors at the time of surgery, cases scoring 4-6 had shorter progression-free survival periods after platinum-based chemotherapy than cases scoring 0-3 (p = 0.012). The cytoskeleton proteins Spectrin αII and ßII contributed to drug resistance by anchoring the GS-Pt complex to the cell membrane, arresting cisplatin activity. Thus spectrin ßII may be a useful predictor of platinum sensitivity in ovarian serous adenocarcinoma.

Enterohaemorrhagic Escherichia coli requires the spectrin cytoskeleton for efficient attachment and pedestal formation on host cells.

Recent work has demonstrated that the spectrin cytoskeleton is a host cell target, exploited during intestinal bacterial disease. Here we show that the highly virulent intestinal pathogen enterohaemorrhagic Escherichia coli (EHEC) is also reliant upon the spectrin cytoskeleton during key pathogenic events. Immunofluorescent microscopy demonstrated that the core components of the spectrin cytoskeleton (spectrin, adducin, and protein 4.1 [p4.1]) are recruited to sites of EHEC attachment and localized at pedestal structures along with the EHEC pedestal specific proteins IRSp53 and IRTKS. Further studies involving siRNA-mediated knockdowns of spectrin, adducin, or p4.1 revealed that those proteins are needed for efficient docking of EHEC to host cells, are involved in recruiting IRSp53 to the pedestal and are necessary for pedestal formation. These findings identify the spectrin cytoskeleton as a major host cell cytoskeletal network involved in critical EHEC pathogenic events.

Schwann cell spectrins modulate peripheral nerve myelination.

During peripheral nerve development, Schwann cells ensheathe axons and form myelin to enable rapid and efficient action potential propagation. Although myelination requires profound changes in Schwann cell shape, how neuron-glia interactions converge on the Schwann cell cytoskeleton to induce these changes is unknown. Here, we demonstrate that the submembranous cytoskeletal proteins αII and ßII spectrin are polarized in Schwann cells and colocalize with signaling molecules known to modulate myelination in vitro. Silencing expression of these spectrins inhibited myelination in vitro, and remyelination in vivo. Furthermore, myelination was disrupted in motor nerves of zebrafish lacking αII spectrin. Finally, we demonstrate that loss of spectrin significantly reduces both F-actin in the Schwann cell cytoskeleton and the Nectin-like protein, Necl4, at the contact site between Schwann cells and axons. Therefore, we propose αII and ßII spectrin in Schwann cells integrate the neuron-glia interactions mediated by membrane proteins into the actin-dependent cytoskeletal rearrangements necessary for myelination.

NCAM/spectrin complex disassembly results in PSD perforation and postsynaptic endocytic zone formation.

Mechanisms inducing perforation of the postsynaptic density (PSD) are poorly understood. We show that neural cell adhesion molecule- deficient (NCAM-/-) hippocampal neurons have an abnormally high percentage of synapses with perforated PSDs. The percentage of synapses with perforated PSDs is also increased in wild-type (NCAM+/+) neurons after the disruption of the NCAM/spectrin complex indicating that the NCAM-assembled spectrin cytoskeleton maintains the structural integrity of PSDs. We demonstrate that PSD perforations contain endocytic zones involved in α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) internalization. Induction of long-term potentiation in NCAM+/+ neurons accompanied by insertion of AMPAR into the neuronal cell surface is subsequently followed by formation of perforated synapses and AMPAR endocytosis suggesting that perforation of PSDs is important for membrane homeostasis in activated synapses. In NCAM-/- or NCAM+/+ neurons with dissociated spectrin meshwork, AMPAR endocytosis is enhanced under conditions of basal activity. An abnormally high rate of postsynaptic membrane endocytosis may thus contribute to brain pathologies associated with mutations in NCAM or spectrin.

Correlating the fractional inhibition of caspase-3 in NT2 cells with apoptotic markers using an active-caspase-3 enzyme-linked immunosorbent assay.

A rapid and quantitative method for measuring the activity and fractional inhibition of enzymes within their natural cellular environment remains an unmet need in drug discovery. We describe the use of a nonradioactive quantitative enzyme-linked immunosorbent assay (ELISA) for measuring intracellular caspase activity that is amenable to robotic automation. The ELISA specifically detects active-caspase-3 and was used to correlate the in-cell activity of caspase-3 with the progress of caspase-3-mediated events under varying concentrations of caspase-3 inhibitors in NT2 cells. We examined the cleavage of endogenous substrates (poly(ADP-ribose)polymerase and alphaII-spectrin), the extent of DNA fragmentation, and the autocatalytic removal of the caspase-3 prodomain as markers of caspase-3 activity. To impart inhibition of the downstream markers, a greater level of caspase-3 inhibition was required. Although the functional markers were found not to accurately predict intracellular caspase-3 activity, we found that the inhibition of intracellular caspase-3 was highly correlated (R(2) = 0.96) to the inhibition of DNA fragmentation. Also, by comparing the potency of the different inhibitors against the intracellular enzyme versus the purified enzyme, the effects of inhibitor functional groups on whole-cell activity were addressed.

Identification and functional characterization of protein 4.1R and actin-binding sites in erythrocyte beta spectrin: regulation of the interactions by phosphatidylinositol-4,5-bisphosphate.

The ternary complex of spectrin, F-actin, and protein 4.1R defines the erythrocyte membrane skeletal network, which governs the stability and elasticity of the membrane. It has been shown that both 4.1R and actin bind to the N-terminal region (residues 1-301) of the spectrin beta chain, which contains two calponin homology domains, designated CH1 and CH2. Here, we show that 4.1R also binds to the separate CH1 and CH2 domains. Unexpectedly, truncation of the CH2 domain by its 20 amino acids, corresponding to its N-terminal alpha helix, was found to greatly enhance its binding to 4.1R. The intact N terminus and the CH1 but not the CH2 domain bind to F-actin, but again, deletion of the first 20 amino acids of the latter exposes an actin-binding activity. As expected, the polypeptide 1-301 inhibits the binding of spectrin dimer to actin and formation of the spectrin-actin-4.1R ternary complex in vitro. Furthermore, the binding of 4.1R to 1-301 is greatly enhanced by PIP(2), implying the existence of a regulatory switch in the cell.

Occurrence of lipid receptors inferred from brain and erythrocyte spectrins binding NaOH-extracted and protease-treated neuronal and erythrocyte membranes.

It was previously shown in model systems that brain spectrin binds membrane phospholipids. In the present study, we analysed binding of isolated brain spectrin and red blood cell spectrin to red blood or neuronal membranes which had been treated as follows: (1). extracted with low ionic-strength solution, (2). the above membranes extracted with 0.1 M NaOH, and (3). membranes treated as above, followed by protease treatment and re-extraction with 0.1 M NaOH. It was found that isolated, NaOH-extracted, protease-treated neuronal and red blood cell membranes bind brain and red blood cell spectrin with moderate affinities similar to those obtained in model phospholipid membrane-spectrin interaction experiments. Moreover, this binding was competitively inhibited by liposomes prepared from membrane lipids. The presented results indicate the occurrence of receptor sites for spectrins that are extraction- and protease-resistant, therefore most probably of lipidic nature, in native membranes.

Brain spectrin exerts much stronger effect on anionic phospholipid monolayers than erythroid spectrin.

Red blood cell spectrin and its nonerythroid analogues are linked to integral proteins of the membrane by several skeletal protein receptors, such as ankyrin and protein 4.1 together with p55. However, there are also many reasons for believing that they are insufficient to engender all the properties that characterise the native membrane. Therefore, we are concerned with the mechanism by which brain spectrin interacts with phospholipids of the membrane bilayer. Brain and erythrocyte spectrin were shown previously to bind phospholipid vesicles as well as monolayers prepared from aminophospholipids: phosphatidylethanolamine and phosphatidylserine and their mixtures with phosphatidylcholine (PC). In the present study, it is shown that brain spectrin binds to monolayers prepared from anionic phospholipids, such as phosphatidylinositol (PI), phosphatidic acid (PA), phosphatidyl glycerol, diphosphatidylglycerol, and their mixtures with PC. Brain spectrin injected into the subphase to reach nanomolar concentration induced a substantial increase in the surface pressure of monolayers prepared from the phospholipids and their mixtures mentioned above, possibly by penetrating them. This effect is stronger in the case of monolayers prepared from anionic phospholipids alone and weaker when monolayers were prepared from mixtures with PC. The weakest effect was observed in the case of phosphatidylinositol-4,5-bisphosphate monolayers. An interaction of brain spectrin with monolayers prepared from anionic phospholipids (PI/PC 7:3 and PA/PC 7:3) was inhibited (PI/PC much stronger than PA/PC) by purified erythrocyte ankyrin, which indicates that the binding site for those lipids is located in the beta-subunit, possibly in, or in close proximity of, the ankyrin-binding site. In contrast, erythrocyte spectrin injected into the subphase induced a change in the surface pressure of monolayers prepared from anionic phospholipids, which was equal or smaller than the value of surface pressure change induced by protein without a monolayer. This effect was different from what had been observed previously for monolayers prepared from aminophospholipids and their mixtures with PC, and from the data for nonerythroid spectrin presented here.

Calpain activation and inhibition in organotypic rat hippocampal slice cultures deprived of oxygen and glucose.

It has been suggested that, after ischaemia, activation of proteases such as calpains could be involved in cytoskeletal degradation leading to neuronal cell death. In vivo, calpain inhibitors at high doses have been shown to reduce ischaemic damage and traumatic brain injury, however, the relationship between calpain activation and cell death remains unclear. We have investigated the role of calpain activation in a model of ischaemia based on organotypic hippocampal slice cultures using the appearance of spectrin breakdown products (BDPs) as a measure of calpain I activation. Calpain I activity was detected on Western blot immediately after a 1-h exposure to ischaemia. Up to 4 h post ischaemia, BDPs were found mainly in the CA1 region and appeared before uptake of the vital dye propidium iodide (PI). 24 h after the insult, BDPs were detected extensively in CA1 and CA3 pyramidal cells, all of which was PI-positive. However, there were many more PI-positive cells that did not have BDPs, indicating that the appearance of BDPs does not necessarily accompany ischaemic cell death. Inhibition of BDP formation by the broad-spectrum protease inhibitor leupeptin was not accompanied by any neuroprotective effects. The more specific and more cell-permeant calpain inhibitor MDL 28170 had a clear neuroprotective effect when added after the ischaemic insult. In contrast, when MDL 28170 was present throughout the entire pre- and post-incubation phases, PI labelling actually increased, indicating a toxic effect. These results suggest that calpain activation is not always associated with cell death and that, while inhibition of calpains can be neuroprotective under some conditions, it may not always lead to beneficial outcomes in ischaemia.

Alteration of cell cycle timing and induction of surface instability in starfish blastomeres microinjected with antibodies to spectrin.

Spectrin has been implicated in a variety of different processes during late embryogenesis, after transcription of the zygotic genome has been activated. However, relatively little is known about the role of maternally derived spectrin during the early cleavage divisions that give rise to a multicellular embryo. To investigate the role of spectrin in early development, we have microinjected anti-spectrin antibodies into Patiria miniata starfish embryos to inhibit the activity of the maternal pool of spectrin. Microinjection of affinity-purified anti-spectrin antibody, or low to moderate doses of F(ab) fragments, into one blastomere of a two-cell-stage embryo caused a dose-dependent, progressive increase in the length of the cell cycle compared to the uninjected control blastomere. The progeny of injected blastomeres were unable to participate in the formation of a blastula epithelium, instead forming a loose aggregate of cells that eventually stopped dividing. When division stopped, the cells formed surface protrusions and became motile. At high doses of either whole antibody or F(ab) fragments, cells initiated, but failed to complete, cytokinesis. Blastomeres injected with high doses of F(ab) fragments also failed to reform nuclei and underwent variable periods of cell cycle arrest up to 12 hr. Injected embryos stained with BODIPY-phallacidin exhibited extensive disruption of the cortical actin cytoskeleton. These results support previous studies implicating spectrin in stabilizing the cell surface and maintaining the organization of the cortical cytoskeleton. They further suggest that spectrin is not required for the initiation or contraction of the cleavage furrow, but functions in the completion of cytokinesis. Most surprisingly, however, the results demonstrate that inhibition of spectrin function alters cell cycle timing, suggesting that disruption of the actin cytoskeleton inhibits progression through the cell cycle.

Involvement of spectrin in membrane fusion: induction of fusion in human erythrocyte ghosts by proteolytic enzymes and its inhibition by antispectrin antibody.

In contrast to intact human erythrocytes, human erythrocyte ghosts can be agglutinated but not fused by Sendai virus. Membrane fusion can, however, be induced in virus-agglutinated erythrocyte ghosts by addition of proteolytic enzymes such as trypsin, papain, or Pronase. When erythrocyte ghosts were reacted with antispectrin antiserum, the antiserum inhibited both the induction of fusion and the proteolysis of the membrane spectrin. The correlation between the membrane fusion process and the membrane cytoskeleton is discussed.