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PubMed2006

Met-Independent Hepatocyte Growth Factor-mediated regulation of cell adhesion in human prostate cancer cells.

BACKGROUND: Prostate cancer cells communicate reciprocally with the stromal cells surrounding them, inside the prostate, and after metastasis, within the bone. Each tissue secretes factors for interpretation by the other. One stromally-derived factor, Hepatocyte Growth Factor (HGF), was found twenty years ago to regulate invasion and growth of carcinoma cells. Working with the LNCaP prostate cancer progression model, we found that these cells could respond to HGF stimulation, even in the absence of Met, the only known HGF receptor. The new HGF binding partner we find on the cell surface may help to clarify conflicts in the past literature about Met expression and HGF response in cancer cells. METHODS: We searched for Met or any HGF binding partner on the cells of the PC3 and LNCaP prostate cancer cell models, using HGF immobilized on agarose beads. By using mass spectrometry analyses and sequencing we have identified nucleolin protein as a novel HGF binding partner. Antibodies against nucleolin (or HGF) were able to ameliorate the stimulatory effects of HGF on met-negative prostate cancer cells. Western blots, RT-PCR, and immunohistochemistry were used to assess nucleolin levels during prostate cancer progression in both LNCaP and PC3 models. RESULTS: We have identified HGF as a major signaling component of prostate stromal-conditioned media (SCM) and have implicated the protein nucleolin in HGF signal reception by the LNCaP model prostate cancer cells. Antibodies that silence either HGF (in SCM) or nucleolin (on the cell surfaces) eliminate the adhesion-stimulatory effects of the SCM. Likewise, addition of purified HGF to control media mimics the action of SCM. C4-2, an LNCaP lineage-derived, androgen-independent human prostate cancer cell line, responds to HGF in a concentration-dependent manner by increasing its adhesion and reducing its migration on laminin substratum. These HGF effects are not due to shifts in the expression levels of laminin-binding integrins, nor can they be linked to expression of the known HGF receptor Met, as neither LNCaP nor clonally-derived C4-2 sub-line contain any detectable Met protein. Even in the absence of Met, small GTPases are activated, linking HGF stimulation to membrane protrusion and integrin activation. Membrane-localized nucelolin levels increase during cancer progression, as modeled by both the PC3 and LNCaP prostate cancer progression cell lines. CONCLUSION: We propose that cell surface localized nucleolin protein may function in these cells as a novel HGF receptor. Membrane localized nucleolin binds heparin-bound growth factors (including HGF) and appears upregulated during prostate cancer progression. Antibodies against nucleolin are able to ameliorate the stimulatory effects of HGF on met-negative prostate cancer cells. HGF-nucleolin interactions could be partially responsible for the complexity of HGF responses and met expression reported in the literature.

Cell Adhesion↗
PubMed2006

Translational control of collagen prolyl 4-hydroxylase-alpha(I) gene expression under hypoxia.

Hypoxia is a pro-fibrotic stimulus, which is associated with enhanced collagen synthesis, as well as with augmented collagen prolyl 4-hydroxylase (C-P4H) activity. C-P4H activity is controlled mainly by regulated expression of the alpha C-P4H subunit. In this study we demonstrate that the increased synthesis of C-P4H-alpha(I) protein in human HT1080 fibroblasts under long term hypoxia (36 h, 1% oxygen) is controlled at the translational level. This is mediated by an interaction of RNA-binding protein nucleolin (approximately 64 kDa form) at the 5'- and 3'-untranslated regions (UTR) of the mRNA. The 5'/3'-UTR-dependent mechanism elevates the C-P4H-alpha(I) expression rate 2.3-fold, and participates in a 5.3-fold increased protein level under long term hypoxia. The interaction of nucleolin at the 5'-UTR occurs directly and depends on the existence of an AU-rich element. Statistical evaluation of the approximately 64-kDa nucleolin/RNA interaction studies revealed a core binding sequence, corresponding to UAAAUC or AAAUCU. At the 3'-UTR, nucleolin assembles indirectly via protein/protein interaction, with the help of another 3'-UTR-binding protein, presumably annexin A2. The increased protein level of the approximately 64-kDa nucleolin under hypoxia can be attributed to an autocatalytic cleavage of a high molecular weight nucleolin form, without alterations in nucleolin mRNA concentration. Thus, the alteration of translational efficiency by nucleolin, which occurs through a hypoxia inducible factor independent pathway, is an important step in C-P4H-alpha(I) regulation under hypoxia.

3' Untranslated Regions↗
PubMed1998

IRS pleckstrin homology domains bind to acidic motifs in proteins.

Using a yeast two-hybrid system, we identified several proteins that interact with the PH domains in IRS-1 and IRS-2, including Lon protease, myeloblast protein, and nucleolin. Although the roles of these molecules in insulin action are not yet known, each protein contained an acidic motif that interacted with the PH domain of IRS-2. However, only the acidic motif in nucleolin bound to IRS-1, suggesting that the PH domain in IRS-1 and IRS-2 are not identical. Moreover, synthetic peptides based on the acidic motif in Lon protease and myeloblast protein inhibited the binding of nucleolin to the PH domain of IRS-2 but not to the PH domain of IRS-1, confirming the selectivity of these PH domains. The ability to bind acidic motifs may be a specific function of the PH domain in IRS proteins, because the PH domains in betaARK, phospholipase Cgamma, or spectrin did not bind nucleolin. In 32D cells, nucleolin bound to both IRS-1 and IRS-2, and expression of the acidic motif of nucleolin inhibited insulin-stimulated tyrosine phosphorylation of IRS-1 and IRS-2. These results suggest that the binding of acidic motifs to the PH domain of IRS-1 and IRS-2 disrupts coupling to the activated insulin receptor. Our results are consistent with the hypothesis that the PH domain in the IRS proteins may ordinarily bind acidic peptide motifs in membrane proteins or other acidic membrane elements that couple IRS proteins to activated membrane receptors.

ATP-Dependent Proteases↗
PubMed1996

Structural changes in oocyte nucleoli of Xenopus laevis during oogenesis and meiotic maturation.

Immunoelectron microscopy with anti-nucleolin defined substructures within the multiple nucleoli of biosynthetically active stage II-III oocytes and within the nucleoli of relatively quiescent stage VI oocytes of Xenopus laevis. Dense fibrillar components (DFCs) of nucleoli from stage II-III oocytes consisted of nucleolonemas that radiated from a continuous DFC sheath surrounding fibrillar centers (FCs). Discernible granular regions (GRs) were absent in these same nucleoli. Conversely, stage VI oocyte nucleoli displayed compacted DFCs and prominent GRs. Immunofluorescence microscopy then tracked fibrillarin, nucleolin, and condensed DNA through oogenesis and into progesterone-induced meiotic maturation and nuclear breakdown. In stage II-III oocyte nucleoli, fibrillarin was enriched near the FC-DFC boundaries, while nucleolin was distributed throughout these same DFCs. Both proteins were enriched within the compacted DFCs of stage VI oocyte nucleoli. Staining with (DAPI) 4',6-diamidino-2-phenylindole showed condensed DNA within nucleolar FCs of both stage II-III and stage VI oocyte. Upon nuclear breakdown, we found fibrillarin and nucleolin in small particles and in the surrounding cytoplasm. Although we saw no trace of fibrillarin or nucleolin in nuclear remnants prepared just minutes later, DAPI-stained particles remained within these preparations, thus suggesting that FCs were at least slow to disassemble.

Animals↗
PubMed2000

Nucleolar proteins and nuclear ultrastructure in preimplantation bovine embryos produced in vitro.

The aim of the present investigation was to describe the basic cell biology of the postfertilization activation of rRNA genes using in vitro-produced bovine embryos as a model. We used immunofluorescence confocal laser scanning microscopy and transmission electron microscopy to study nucleolar development in the nuclei of embryos up to the fifth postfertilization cell cycle. During the first cell cycle (1-cell stage), fibrillarin, upstream binding factor (UBF), nucleolin (C23), and RNA polymerase I were localized to distinct foci in the pronuclei, and, ultrastructurally, compact spherical fibrillar masses were the most prominent pronuclear finding. During the second cell cycle (2-cell stage), the findings were similar except for a lack of nucleolin and RNA polymerase I labeling. During the third cell cycle (4-cell stage), fibrillarin, UBF, nucleophosmin, and nucleolin were localized to distinct foci. Ultrastructurally, spherical fibrillar masses that developed a central vacuole over the course of the cell cycle were observed. Early in the fourth cell cycle (8-cell stage), fibrillarin, nucleophosmin, and nucleolin were localized to small bodies that with time developed a central vacuole. UBF and topoisomerase I were localized to clusters of small foci. Ultrastructurally, spherical fibrillar masses with a large eccentric vacuole and later small peripheral vacuoles were seen. Late in the fourth cell cycle, nucleophosmin and nucleolin were localized to large shell-like bodies; and fibrillarin, UBF, topoisomerase I, and RNA polymerase I were localized to clusters of small foci. Ultrastructurally, a presumptive dense fibrillar component (DFC) and fibrillar centers (FCs) were observed peripherally in the vacuolated spherical fibrillar masses. Subsequently, the presumptive granular component (GC) gradually became embedded in the substance of this entity, resulting in the formation of a fibrillo-granular nucleolus. During the fifth cell cycle (16-cell stage), a spherical fibrillo-granular nucleolus developed from the start of the cell cycle. In conclusion, the nucleolar protein compartment in in vitro-produced preimplantation bovine embryos is assembled over several cell cycles. In particular, RNA polymerase I and topoisomerase I are detected for the first time late during the fourth embryonic cell cycle, which coincides with the first recognition of the DFC, FCs, and GC at the ultrastructural level.

Animals↗
PubMed2001

Immunolocalization of nucleolar proteins during bovine oocyte growth, meiotic maturation, and fertilization.

During the growth phase of the bovine oocyte transcripts, polypeptides and ribosomes are accumulated in the oocyte to drive and sustain future meiotic maturation, fertilization, and early embryonic development. The oocyte also furnishes the early embryo with the components required to establish a functional transcriptionally active nucleolus at the time of maternal embryonic transition. The aim of the present study was to describe the behavior of key components of the nucleolus. The temporal localization of nucleolar proteins fibrillarin, nucleophosmin, nucleolin, RNA polymerase I (RNA pol I), topoisomerase I, upstream binding factor (UBF), and coilin 5P10 was investigated in growing and fully grown immature bovine oocytes during in vitro maturation and during the first postfertilization cell cycle using whole-mount immunocytochemistry and confocal microscopy. During the oocyte growth phase, fibrillarin, nucleophosmin, nucleolin, RNA pol I, and UBF were localized to the oocyte nucleolus. On completion of the growth phase, nucleolin and nucleophosmin appeared to migrate to the periphery of the nucleolus and into the nucleoplasm, and the proportion of oocytes displaying RNA pol I localization had decreased. Topoisomerase I was not detected at any stage. Fibrillarin appeared to be localized to large foci within the nucleolus and/or nucleoplasm. Nucleophosmin and nucleolin labeling was characterized by a homogeneous signal over the nucleolus. RNA pol I and UBF were characterized by the localization of the antibodies to individual or clustered foci in the nucleolus and/or nucleoplasm. Following oocyte nucleus breakdown (ONBD), the proteins appeared to disperse into the cytoplasm. All proteins were undetectable during meiotic maturation and were not relocalized until 5-10 h postinsemination (hpi). UBF was localized to the fertilizing sperm head of most zygotes at 5 hpi. By 10 hpi, all proteins were detected in most oocytes displaying two pronuclei. Nucleolar protein localization was exclusive to or more abundant in one pronucleus up to 20 hpi; thereafter, the pattern was more evenly distributed. Fibrillarin, nucleophosmin, nucleolin, UBF, and Pol I are present in the nuclei of growing and fully grown bovine oocytes until ONBD. They reappear at the late telophase stage of meiosis II and continue to be present up to the first mitotic division of embryo development.

Animals↗
PubMed2002

Nuclear targeting by the growth factor midkine.

Ligand-receptor internalization has been traditionally regarded as part of the cellular desensitization system. Low-density lipoprotein receptor-related protein (LRP) is a large endocytosis receptor with a diverse array of ligands. We recently showed that LRP binds heparin-binding growth factor midkine. Here we demonstrate that LRP mediates nuclear targeting by midkine and that the nuclear targeting is biologically important. Exogenous midkine reached the nucleus, where intact midkine was detected, within 20 min. Midkine was not internalized in LRP-deficient cells, whereas transfection of an LRP expression vector restored midkine internalization and subsequent nuclear translocation. Internalized midkine in the cytoplasm bound to nucleolin, a nucleocytoplasmic shuttle protein. The midkine-binding sites were mapped to acidic stretches in the N-terminal domain of nucleolin. When the nuclear localization signal located next to the acidic stretches was deleted, we found that the mutant nucleolin not only accumulated in the cytoplasm but also suppressed the nuclear translocation of midkine. By using cells that overexpressed the mutant nucleolin, we further demonstrated that the nuclear targeting was necessary for the full activity of midkine in the promotion of cell survival. This study therefore reveals a novel role of LRP in intracellular signaling by its ligand and the importance of nucleolin in this process.

Active Transport, Cell Nucleus↗
PubMed2003

Activation of Epstein-Barr virus/C3d receptor (gp140, CR2, CD21) on human cell surface triggers pp60src and Akt-GSK3 activities upstream and downstream to PI 3-kinase, respectively.

We previously demonstrated that CR2 activation on human B lymphocyte surface specifically triggered tyrosine phosphorylation of the 95-kDa nucleolin, this leading to its binding on SH2 domains of p85 sub-unit of PI 3-kinase and to activation of this enzyme. The specificity of CR2 pathway was clearly demonstrated as neither CD19 nor BCR could induce tyrosine phosphorylation of nucleolin in normal B lymphocytes. These data led us to investigate herein additional molecular events, which were triggered by CR2 activation, upstream and downstream to PI 3-kinase activation. Upstream, we demonstrated that pp60src, a tyrosine kinase of the src family, was involved in tyrosine phosphorylation of nucleolin, while syk tyrosine kinase was not. We also demonstrated a direct protein-protein interaction of pp60src with nucleolin in a CR2-dependent and CD19-independent pathway. Downstream, we demonstrated that CR2 activation also triggered Akt and GSK3 enzyme activation, this pathway being under the control of pp60src tyrosine kinase activation. These regulatory functions of activated CR2 were specific as independent of syk tyrosine kinase and of CD19 and BCR activation. Thus, CR2 activation recruits a specific mechanism to activate PI 3-kinase and its subsequent pathways, this mechanism being different to those recruited by CD19 and BCR.

Antigens, CD19↗
PubMed1997

Small nucleolar RNAs and nucleolar proteins in Xenopus anucleolate embryos.

We investigated the presence and localization, in the cells of anucleolate mutant embryos of Xenopus laevis, of three representative small nucleolar RNAs (snoRNAs), U3, U15 and U17, and of two nucleolar proteins, nucleolin and fibrillarin. The levels of the three snoRNAs in the anucleolate mutant are the same as in normal embryos, in contrast to 5S RNA and ribosomal proteins. In situ hybridization showed that, in the absence of fully organized nucleoli, the three RNAs are diffusely distributed in the nucleus and partly associated with a number of small structures. Nucleolin and fibrillarin are also present in the anucleolate embryos as in normal embryos, although there is less nucleolin mRNA in the former. The two nucleolar proteins were localized by immunofluorescence microscopy. Fibrillarin, similar to its associated U3 and U15 snoRNAs, is diffusely distributed in the anucleolate nucleus and is partly associated with small structures, probably prenucleolar bodies and pseudonucleoli. Nucleolin also appears diffusely distributed in the nucleus with some spots of higher concentration, but with a different pattern with respect to fibrillarin.

Animals↗
PubMed2000

Initiation of nucleolar assembly is independent of RNA polymerase I transcription.

This report examines the distribution of an RNA polymerase I transcription factor (upstream binding factor; UBF), pre-rRNA processing factors (nucleolin and fibrillarin), and pre-rRNAs throughout mitosis and postmitotic nucleologenesis in HeLa cells. The results demonstrate that nucleolin, fibrillarin, and pre-rRNAs synthesized at G2/M phase of the previous cell cycle are directly recruited to UBF-associated nucleolar organizer regions (NORs) early in telophase before chromosome decondensation. Unlike the fusion of prenucleolar bodies to the nucleoli, this early recruitment of processing factors and pre-rRNAs is independent of RNA polymerase I transcription. In the absence of polymerase I transcription, the initial localization of nucleolin, fibrillarin, and pre-rRNAs to UBF-associated NORs generates segregated mininucleoli that are similar to the larger ones observed in interphase cells grown under the same conditions. Pre-rRNAs are juxtaposed to UBF-nucleolin-fibrillarin caps that may represent the segregated nucleoli observed by electron microscopy. These findings lead to a revised model of nucleologenesis. We propose that nucleolar formation at the end of mitosis results from direct recruitment of processing factors and pre-rRNAs to UBF-associated NORs before or at the onset of rDNA transcription. This is followed by fusion of prepackaged prenucleolar bodies into the nucleolus. Pre-ribosomal ribonucleoproteins synthesized in the previous cell cycle may contribute to postmitotic nucleologenesis.

Cell Cycle↗
PubMed2000

Role for nucleolin/Nsr1 in the cellular localization of topoisomerase I.

Nucleolin functions in ribosome biogenesis and contains an acidic N terminus that binds nuclear localization sequences. In previous work we showed that human nucleolin associates with the N-terminal region of human topoisomerase I (Top1). We have now mapped the topoisomerase I interaction domain of nucleolin to the N-terminal 225 amino acids. We also show that the Saccharomyces cerevisiae nucleolin ortholog, Nsr1p, physically interacts with yeast topoisomerase I, yTop1p. Studies of isogenic NSR1(+) and Deltansr1 strains indicate that NSR1 is important in determining the cellular localization of yTop1p. Moreover, deletion of NSR1 reduces sensitivity to camptothecin, an antineoplastic topoisomerase I inhibitor. By contrast, Deltansr1 cells are hypersensitive to the topoisomerase II-targeting drug amsacrine. These findings indicate that nucleolin/Nsr1 is involved in the cellular localization of Top1 and that this localization may be important in determining sensitivity to drugs that target topoisomerases.

Amsacrine↗
PubMed2004

Evidence for an S-phase checkpoint regulating DNA replication after heat shock: a review.

Exposure of cells to heat inhibits a number of nuclear activities associated with semi-conservative replication of DNA including the incorporation of radiolabelled precursors into acid-insoluble DNA, the initiation of new replicons, the elongation of the DNA fibre at the replication fork, the synthesis and deposition of new histones into chromatin and the reorganization of nascent DNA into mature chromatin. These effects are likely to underlie the heat sensitivity of S-phase cells and may contribute to the radiosensitization observed in this phase of the cell cycle. While some of these effects may be explained as 'passive' consequences of heat-induced damage on chromatin structures experiments reviewed here point to the activation of a checkpoint as a contributing factor to the observed inhibition of DNA replication. Activation of a heat responsive S-phase checkpoint targets the activity of RPA via interaction with nucleolin. Nucleolin, a major nucleolar protein, is found normally sequestered in the nucleolus. Exposure of cells to heat causes a rapid translocation of nucleolin from the nucleolus into the nucleoplasm that enables RPA/nucleolin interaction. This interaction inhibits functions of RPA associated with the initiation of DNA replication and contributes to the immediate inhibition of DNA synthesis observed after heat shock. The results suggest that the nucleolus serves as a sequestration centre for the temporary inactivation of regulatory molecules, such as nucleolin, capable of regulating essential cellular functions after heat shock. It is speculated that this regulatory process is integrated in the network of responses that determine cell sensitivity to heat and that it may be involved in heat radiosensitization to killing as well.

Animals↗
PubMed1998

hnRNP C increases amyloid precursor protein (APP) production by stabilizing APP mRNA.

We have previously shown that heterogeneous nuclear ribonucleoprotein C (hnRNP C) and nucleolin bound specifically to a 29 nt sequence in the 3'-untranslated region of amyloid precursor protein (APP) mRNA. Upon activation of peripheral blood mononuclear cells, hnRNP C and nucleolin acquired APP mRNA binding activity, concurrent with APP mRNA stabilization. These data suggested that the regulated interaction of hnRNP C and nucleolin with APP mRNA controlled its stability. Here we have directly examined the role of the cis element and trans factors in the turnover and translation of APP mRNA in vitro . In a rabbit reticulocyte lysate (RRL) translation system, a mutant APP mRNA lacking the 29 nt element was 3-4-fold more stable and synthesized 2-4-fold more APP as wild-type APP mRNA. Therefore, the 29 nt element functioned as an APP mRNA destabilizer. RNA gel mobility shift assays with the RRL suggested the presence of endogenous nucleolin, but failed to show hnRNP C binding activity. However, wild-type APP mRNA was stabilized and coded for 6-fold more APP when translated in an RRL system supplemented with exogenous active hnRNP C. Control mRNAs lacking the 29 nt element were unaffected by hnRNP C supplementation. Therefore, occupancy of the 29 nt element by hnRNP C stabilized APP mRNA and enhanced its translation.

Amyloid beta-Protein Precursor↗