89 research outputs found
Transferrin-polycation conjugates as carriers for DNA uptake into cells.
We have developed a high-efficiency nucleic acid delivery system that uses receptor-mediated endocytosis to carry DNA macromolecules into cells. We accomplished this by conjugating the iron-transport protein transferrin to polycations that bind nucleic acids. Human transferrin, as well as the chicken homologue conalbumin, has been covalently linked to the small DNA-binding protein protamine or to polylysines of various sizes through a disulfide linkage. These modified transferrin molecules maintain their ability to bind their cognate receptor and to mediate efficient iron transport into the cell. The transferrin-polycation molecules form electrophoretically stable complexes with double-stranded DNA, single-stranded DNA, and modified RNA molecules independent of nucleic acid size (from short oligonucleotides to DNA of 21 kilobase pairs). When complexes of transferrin-polycation and a bacterial plasmid DNA containing the gene for Photinus pyralis luciferase are supplied to eukaryotic cells, high-level expression of the luciferase gene occurs, demonstrating transferrin receptor-mediated endocytosis and expression of the imported DNA. We refer to this delivery system as "transferrinfection.
E-cadherin regulates cell growth by modulating proliferation-dependent β-catenin transcriptional activity
β-Catenin is essential for E-cadherin–mediated cell adhesion in epithelial cells, but it also forms nuclear complexes with high mobility group transcription factors. Using a mouse mammary epithelial cell system, we have shown previously that conversion of epithelial cells to a fibroblastoid phenotype (epithelial-mesenchymal transition) involves downregulation of E-cadherin and upregulation of β-catenin transcriptional activity. Here, we demonstrate that transient expression of exogenous E-cadherin in both epithelial and fibroblastoid cells arrested cell growth or caused apoptosis, depending on the cellular E-cadherin levels. By expressing E-cadherin subdomains, we show that the growth-suppressive effect of E-cadherin required the presence of its cytoplasmic β-catenin interaction domain and/or correlated strictly with the ability to negatively interfere with β-catenin transcriptional activity. Furthermore, coexpression of β-catenin or lymphoid enhancer binding factor-1 or T cell factor 3 with E-cadherin rescued β-catenin transcriptional activity and counteracted E-cadherin–mediated cell cycle arrest. Stable expression of E-cadherin in fibroblastoid cells decreased β-catenin activity and reduced cell growth. Since proliferating cells had a higher β-catenin activity than G1 phase–arrested or contact-inhibited cells, we conclude that β-catenin transcriptional activity is essential for cell proliferation and can be controlled by E-cadherin in a cell adhesion-independent manner
Igbp1 is part of a positive feedback loop in stem cell factor–dependent, selective mRNAtranslation initiation inhibiting erythroid differentiation
The authors thank Dr Victor de Jager for assistance with the Rosetta
Resolver software; Dr Ivo Touw for many fruitful discussions and
critical reading of the manuscript; Liu Wing for technical assistance;
Drs Peter Seither, Andreas Weith (Boehringer Ingelheim,
Biberach, Germany), Helmuth Dolznig, Thomas Waerner, and
Sandra Pilat (IMP, Vienna, Austria) for mRNA profiling of
erythroblasts, of which the complete data will be published
elsewhere; Dr Bart Aarts (Erasmus MC, Rotterdam, The Netherlands)
for assistance in confocal scanning microscopy; Dr David Brautigan
(University of Virginia, Charlottesville) for anti-Igbp1 antibodies;
Dr Manfred Boehm (National Institutes of Health/National
Heart, Lung, and Blood Institute, Bethesda, MD) for anti-Uhmk1
antibodies; and Ortho-Biotech (Tilburg, The Netherlands) for their
kind gift of Eprex (erythropoietin).Stem cell factor (SCF)–induced activation
of phosphoinositide-3-kinase (PI3K) is required
for transient amplification of the
erythroblast compartment. PI3K stimulates
the activation of mTOR (target of
rapamycin) and subsequent release of
the cap-binding translation initiation factor
4E (eIF4E) from the 4E-binding protein
4EBP, which controls the recruitment of
structured mRNAs to polysomes. Enhanced
expression of eIF4E renders proliferation
of erythroblasts independent of
PI3K. To investigate which mRNAs are
selectively recruited to polysomes, we
compared SCF-dependent gene expression
between total and polysome-bound
mRNA. This identified 111 genes primarily
subject to translational regulation. For
8 of 9 genes studied in more detail, the
SCF-induced polysome recruitment of
transcripts exceeded 5-fold regulation and
was PI3K-dependent and eIF4E-sensitive,
whereas total mRNA was not affected by
signal transduction. One of the targets,
Immunoglobulin binding protein 1 (Igbp1),
is a regulatory subunit of protein phosphatase
2A (Pp2a) sustaining mTOR signaling.
Constitutive expression of Igbp1
impaired erythroid differentiation, maintained
4EBP and p70S6k phosphorylation,
and enhanced polysome recruitment
of multiple eIF4E-sensitive mRNAs.
Thus, PI3K-dependent polysome recruitment
of Igbp1 acts as a positive feedback
mechanism on translation initiation underscoring
the important regulatory role of
selectivemRNArecruitment to polysomes
in the balance between proliferation and
maturation of erythroblasts. (Blood. 2008;
112:2750-2760)peer-reviewe
Ras and TGFβ cooperatively regulate epithelial cell plasticity and metastasis: dissection of Ras signaling pathways
Multistep carcinogenesis involves more than six discrete events also important in normal development and cell behavior. Of these, local invasion and metastasis cause most cancer deaths but are the least well understood molecularly. We employed a combined in vitro/in vivo carcinogenesis model, that is, polarized Ha-Ras–transformed mammary epithelial cells (EpRas), to dissect the role of Ras downstream signaling pathways in epithelial cell plasticity, tumorigenesis, and metastasis. Ha-Ras cooperates with transforming growth factor β (TGFβ) to cause epithelial mesenchymal transition (EMT) characterized by spindle-like cell morphology, loss of epithelial markers, and induction of mesenchymal markers. EMT requires continuous TGFβ receptor (TGFβ-R) and oncogenic Ras signaling and is stabilized by autocrine TGFβ production. In contrast, fibroblast growth factors, hepatocyte growth factor/scatter factor, or TGFβ alone induce scattering, a spindle-like cell phenotype fully reversible after factor withdrawal, which does not involve sustained marker changes. Using specific inhibitors and effector-specific Ras mutants, we show that a hyperactive Raf/mitogen-activated protein kinase (MAPK) is required for EMT, whereas activation of phosphatidylinositol 3-kinase (PI3K) causes scattering and protects from TGFβ-induced apoptosis. Hyperactivation of the PI3K pathway or the Raf/MAPK pathway are sufficient for tumorigenesis, whereas EMT in vivo and metastasis required a hyperactive Raf/MAPK pathway. Thus, EMT seems to be a close in vitro correlate of metastasis, both requiring synergism between TGFβ-R and Raf/MAPK signaling
NF-κB is essential for epithelial-mesenchymal transition and metastasis in a model of breast cancer progression
Analysis of Cd44-Containing Lipid Rafts: Recruitment of Annexin II and Stabilization by the Actin Cytoskeleton
CD44, the major cell surface receptor for hyaluronic acid (HA), was shown to localize to detergent-resistant cholesterol-rich microdomains, called lipid rafts, in fibroblasts and blood cells. Here, we have investigated the molecular environment of CD44 within the plane of the basolateral membrane of polarized mammary epithelial cells. We show that CD44 partitions into lipid rafts that contain annexin II at their cytoplasmic face. Both CD44 and annexin II were released from these lipid rafts by sequestration of plasma membrane cholesterol. Partition of annexin II and CD44 to the same type of lipid rafts was demonstrated by cross-linking experiments in living cells. First, when CD44 was clustered at the cell surface by anti-CD44 antibodies, annexin II was recruited into the cytoplasmic leaflet of CD44 clusters. Second, the formation of intracellular, submembranous annexin II–p11 aggregates caused by expression of a trans-dominant mutant of annexin II resulted in coclustering of CD44. Moreover, a frequent redirection of actin bundles to these clusters was observed. These basolateral CD44/annexin II–lipid raft complexes were stabilized by addition of GTPγS or phalloidin in a semipermeabilized and cholesterol-depleted cell system. The low lateral mobility of CD44 in the plasma membrane, as assessed with fluorescent recovery after photobleaching (FRAP), was dependent on the presence of plasma membrane cholesterol and an intact actin cytoskeleton. Disruption of the actin cytoskeleton dramatically increased the fraction of CD44 which could be recovered from the light detergent-insoluble membrane fraction. Taken together, our data indicate that in mammary epithelial cells the vast majority of CD44 interacts with annexin II in lipid rafts in a cholesterol-dependent manner. These CD44-containing lipid microdomains interact with the underlying actin cytoskeleton
FoxO3a regulates erythroid differentiation and induces BTG1, an activator of protein arginine methyl transferase 1
Erythropoiesis requires tight control of expansion, maturation, and survival of erythroid progenitors. Because activation of phosphatidylinositol-3-kinase (PI3K) is required for erythropoietin/stem cell factor–induced expansion of erythroid progenitors, we examined the role of the PI3K-controlled Forkhead box, class O (FoxO) subfamily of Forkhead transcription factors. FoxO3a expression and nuclear accumulation increased during erythroid differentiation, whereas untimely induction of FoxO3a activity accelerated differentiation of erythroid progenitors to erythrocytes. We identified B cell translocation gene 1 (BTG1)/antiproliferative protein 2 as a FoxO3a target gene in erythroid progenitors. Promoter studies indicated BTG1 as a direct target of FoxO3a. Expression of BTG1 in primary mouse bone marrow cells blocked the outgrowth of erythroid colonies, which required a domain of BTG1 that binds protein arginine methyl transferase 1. During erythroid differentiation, increased arginine methylation coincided with BTG1 expression. Concordantly, inhibition of methyl transferase activity blocked erythroid maturation without affecting expansion of progenitor cells. We propose FoxO3a-controlled expression of BTG1 and subsequent regulation of protein arginine methyl transferase activity as a novel mechanism controlling erythroid expansion and differentiation
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