An Endothelial Cell Genetic Screen Identifies the GTPase Rem2 as a Suppressor of p19ARF Expression That Promotes Endothelial Cell Proliferation and Angiogenesis
2007; Elsevier BV; Volume: 283; Issue: 7 Linguagem: Inglês
10.1074/jbc.m707438200
ISSN1083-351X
AutoresRuben Bierings, Miguel Beato, Michael J. Edel,
Tópico(s)Signaling Pathways in Disease
ResumoAngiogenesis requires an increase in endothelial cell proliferation to support an increase in mass of blood vessels. We designed an in vitro endothelial cell model to functionally screen for genes that regulate endothelial cell proliferation. A gain of function screen for genes that bypass p53 endothelial cell arrest identified Rem2, a Ras-like GTPase. We show that ectopic Rem2 suppresses p14ARF (human) or p19ARF (mouse) expression that leads to increased endothelial cell proliferation. Conversely, loss of ectopic Rem2 by RNA interference restores p19ARF expression in endothelial cells. We further show that Rem2-interacting 14-3-3 proteins are involved in the cell localization of Rem2, regulation of p19ARF expression, and endothelial cell proliferation. Finally, we demonstrate using the RIP1 tag2 mouse model of pancreatic disease that Rem2 is up-regulated in endothelial cells of stage IV disease. The data unravel a possible molecular mechanism for Rem2-induced angiogenesis and suggests Rem2 as a potential novel target for treating pathological angiogenesis. Angiogenesis requires an increase in endothelial cell proliferation to support an increase in mass of blood vessels. We designed an in vitro endothelial cell model to functionally screen for genes that regulate endothelial cell proliferation. A gain of function screen for genes that bypass p53 endothelial cell arrest identified Rem2, a Ras-like GTPase. We show that ectopic Rem2 suppresses p14ARF (human) or p19ARF (mouse) expression that leads to increased endothelial cell proliferation. Conversely, loss of ectopic Rem2 by RNA interference restores p19ARF expression in endothelial cells. We further show that Rem2-interacting 14-3-3 proteins are involved in the cell localization of Rem2, regulation of p19ARF expression, and endothelial cell proliferation. Finally, we demonstrate using the RIP1 tag2 mouse model of pancreatic disease that Rem2 is up-regulated in endothelial cells of stage IV disease. The data unravel a possible molecular mechanism for Rem2-induced angiogenesis and suggests Rem2 as a potential novel target for treating pathological angiogenesis. Angiogenesis, the formation of new blood vessels, is a complex process that involves many steps including endothelial cell proliferation, migration and invasion, tube formation, and vessel maturation. Angiogenesis is involved in disease states such as arthritis and atherosclerosis, and is particularly important for the growth, invasion, and metastatic spread of tumors (1Weidner N. Folkman J. Important Adv. Oncol. 1996; : 167-190PubMed Google Scholar, 2Folkman J. N. Engl. J. Med. 1989; 320: 1211-1212Crossref PubMed Scopus (149) Google Scholar). Although the regulation of endothelial cell proliferation is therefore of obvious importance, very little is known about cell cycle control in endothelial cells. The Ink4a-Arf locus encodes two tumor suppressor proteins, p16INK4a and p19ARF (p14 ARF in humans) that up-regulate the activities of the retinoblastoma protein (Rb) 3The abbreviations used are: Rbretinoblastoma proteinRGKRem/Rad/Gem/KirHUVEChuman umbilical vein endothelial cellHMEChuman microvascular endothelial cellGFPgreen fluorescent proteinRNAiRNA interferenceHAhemagglutininWTwild typeshRNAshort hairpin RNACMVcytomegalovirusCaMcalmodulin.3The abbreviations used are: Rbretinoblastoma proteinRGKRem/Rad/Gem/KirHUVEChuman umbilical vein endothelial cellHMEChuman microvascular endothelial cellGFPgreen fluorescent proteinRNAiRNA interferenceHAhemagglutininWTwild typeshRNAshort hairpin RNACMVcytomegalovirusCaMcalmodulin. and the p53 transcription factor, respectively (3Sherr C.J. McCormick F. Cancer Cell. 2002; 2: 103-112Abstract Full Text Full Text PDF PubMed Scopus (1301) Google Scholar). The p16INK4a protein inhibits the activity of cyclin D-dependent kinases, thereby maintaining Rb in its hypophosphorylated, growth-suppressive state (3Sherr C.J. McCormick F. Cancer Cell. 2002; 2: 103-112Abstract Full Text Full Text PDF PubMed Scopus (1301) Google Scholar). p19ARF antagonizes Mdm2 activity, resulting in a p53 transcriptional response that leads to cell cycle arrest or apoptosis. Loss of p16INK4a or p19ARF function is a critical event for tumor promotion as evidenced by extinguished expression of the p16INK4a and p19ARF protein in a variety of tumors (4Ruas M. Peters G. Biochim. Biophys. Acta. 1998; 1378: F115-F177Crossref PubMed Scopus (873) Google Scholar, 5Esteller M. Corn P.G. Baylin S.B. Herman J.G. Cancer Res. 2001; 61: 3225-3229PubMed Google Scholar). Interestingly, p19ARF loss affects the normal development of the eyes of newborn mice, where persistence of the hyaloid vasculature in the vitreous results in destruction of both the lens and neuroretina, directly implicating p19ARF in regulation of pathological angiogenesis (6McKeller R.N. Fowler J.L. Cunningham J.J. Warner N. Smeyne R.J. Zindy F. Skapek S.X. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 3848-3853Crossref PubMed Scopus (107) Google Scholar, 7Silva R.L. Thornton J.D. Martin A.C. Rehg J.E. Bertwistle D. Zindy F. Skapek S.X. EMBO J. 2005; 24: 2803-2814Crossref PubMed Scopus (48) Google Scholar). retinoblastoma protein Rem/Rad/Gem/Kir human umbilical vein endothelial cell human microvascular endothelial cell green fluorescent protein RNA interference hemagglutinin wild type short hairpin RNA cytomegalovirus calmodulin. retinoblastoma protein Rem/Rad/Gem/Kir human umbilical vein endothelial cell human microvascular endothelial cell green fluorescent protein RNA interference hemagglutinin wild type short hairpin RNA cytomegalovirus calmodulin. Rem2 is a recently identified member of the Rem/Rad/Gem/Kir (RGK) family of Ras-related GTPases that share structural features that are different from other Ras-related proteins (8Finlin B.S. Shao H. Kadono-Okuda K. Guo N. Andres D.A. Biochem. J. 2000; 347: 223-231Crossref PubMed Scopus (88) Google Scholar, 9Finlin B.S. Mosley A.L. Crump S.M. Correll R.N. Ozcan S. Satin J. Andres D.A. J. Biol. Chem. 2005; 280: 41864-41871Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). These include non-conservative amino acid substitutions within regions known to be involved in guanine nucleotide binding and hydrolysis, extended N and C termini, and a conserved C-terminal motif thought to mediate membrane association but lacking a prenylation site present in other Ras-like molecules (10Del Villar K. Dorin D. Sattler I. Urano J. Poullet P. Robinson N. Mitsuzawa H. Tamanoi F. Biochem. Soc. Trans. 1996; 24: 709-713Crossref PubMed Scopus (30) Google Scholar). Two important functions of RGK proteins are the regulation of voltage-gated Ca2+ channel activity and cell shape associated with angiogenesis (11Finlin B.S. Crump S.M. Satin J. Andres D.A. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 14469-14474Crossref PubMed Scopus (164) Google Scholar, 12Piddini E. Schmid J.A. de Martin R. Dotti C.G. EMBO J. 2001; 20: 4076-4087Crossref PubMed Scopus (66) Google Scholar, 13Beguin P. Mahalakshmi R.N. Nagashima K. Cher D.H. Kuwamura N. Yamada Y. Seino Y. Hunziker W. Biochem. J. 2005; 390: 67-75Crossref PubMed Scopus (62) Google Scholar, 14Pan J.Y. Fieles W.E. White A.M. Egerton M.M. Silberstein D.S. J. Cell Biol. 2000; 149: 1107-1116Crossref PubMed Scopus (38) Google Scholar). RGK proteins interact with 14-3-3 and calmodulin and Gem regulates endothelial shape changes required for angiogenesis (12Piddini E. Schmid J.A. de Martin R. Dotti C.G. EMBO J. 2001; 20: 4076-4087Crossref PubMed Scopus (66) Google Scholar, 13Beguin P. Mahalakshmi R.N. Nagashima K. Cher D.H. Kuwamura N. Yamada Y. Seino Y. Hunziker W. Biochem. J. 2005; 390: 67-75Crossref PubMed Scopus (62) Google Scholar). Recently it has been shown that 14-3-3, together with calmodulin, regulates the subcellular distribution of Rem2 between the cytoplasm and the nucleus and this distribution has been correlated to cell shape changes (12Piddini E. Schmid J.A. de Martin R. Dotti C.G. EMBO J. 2001; 20: 4076-4087Crossref PubMed Scopus (66) Google Scholar, 13Beguin P. Mahalakshmi R.N. Nagashima K. Cher D.H. Kuwamura N. Yamada Y. Seino Y. Hunziker W. Biochem. J. 2005; 390: 67-75Crossref PubMed Scopus (62) Google Scholar, 15Beguin P. Mahalakshmi R.N. Nagashima K. Cher D.H. Takahashi A. Yamada Y. Seino Y. Hunziker W. J. Cell Sci. 2005; 118: 1923-1934Crossref PubMed Scopus (69) Google Scholar). However, given past research efforts, little is known about the cell function of the RGK family of GTPases, particularly Rem2. Here we show a novel function for Rem2 as a suppressor of p19ARF transcription, partially dependent on 14-3-3 protein binding to promote accelerated endothelial cell proliferation. Furthermore, we show that ectopic Rem2 promotes angiogenesis in vitro and is expressed in endothelial cells of the RIP1 tag2 mouse model of pancreatic disease. Cell Culture, Transfection, and Infection–Large T SV40 temperature-sensitive endothelial cells (ts T endothelial cells) were subcloned from a polyclonal population of brain capillary endothelial cell line, derived from H-2Kb-tsA58 transgenic mice, and display endothelial cell-specific characters, such as expression of von Willebrand factor and uptake of acetylated low density lipoprotein (16Kanda S. Landgren E. Ljungstrom M. Claesson-Welsh L. Cell Growth & Differ. 1996; 7: 383-395PubMed Google Scholar). Endothelial cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Twenty clones were expanded and 3 clones selected based on the lowest number of background colonies that grew at 39 °C. Experiments were performed with clones 5 and 12 (LTEC5 and LTEC12: large T endothelial Cell 5 and 12) that have virtually no background growth at 39 °C (see Fig. 1). The functional screen was carried out with clone LTEC5, which showed lowest background in repetitive experiments, and genes discovered were later retested in mouse ts T endothelial cells LTEC5 and LTEC12. All results are shown with LTEC5 cell line. Ecotropic retroviral supernatants were produced by transfection of genes into Phoenix packaging cells by calcium-phosphate precipitation. Cells were cultured in Dulbecco's modified Eagle's medium supplemented with 8% fetal calf serum at 37 °C. Forty-eight hours post-transfection, the tissue culture medium was filtered through a 0.45-μm filter and added to target cells. High titer retroviral library supernatants derived from mouse whole brain (Clontech) were used to infect 2 × 106 ts T endothelial cells. Twenty-four hours after infection, cells were plated at a density of ∼1 × 105 cells per 10-cm dish and 48 h after infection the cells were shifted to 39 °C (the non-permissive temperature for the transforming SV40 T antigen). Colonies of cells appeared only in the library-infected populations. These colonies were picked, expanded, and pro-viral inserts identified by re-cloning into library vector (pLIB) and identified by sequencing. To analyze whether the rescue was due to expression of a retroviral library-derived cDNA, a second round with re-cloned library cDNA was performed. Primary HUVECs were isolated from umbilical veins and cultured in medium containing RPMI 1640 and M199 (1:1), 20% human serum, 100 units/ml penicillin, 100 mg/ml streptomycin, and 33 mg/ml glutamine. 500,000 HUVECs at passage 2-3 were electroporated with 9 μg of RGK expressing vector by a previously described method (17Agami R. Bernards R. Cell. 2000; 102: 55-66Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar). Cells were allowed to recover and 200,000 cells plated on fibronectin-coated plates to determine growth rate after 3 days. Immunohistochemistry was performed concurrently as described below. Human microvascular endothelial cells (HMEC-1) were cultured in Dulbecco's modified Eagle's medium with 10% serum on gelatin-coated plates. Ecotropic receptor was electroporated into HMEC-1 cells following a described method and clones selected with Zeomycin. Stable HMEC-1 ecotropic receptor expressing cells were then infected with high titer GFP, Rem2HA, or Ras v12 virus and injected into mice for Matrigel plug angiogenesis assay (see below). cDNA and RNAi Constructs–For overexpression studies we used the TBX3, pBabe Zeo Ecotropic Receptor, LZRS-p19ARF (RED), pBabe-Ras v12, and GFP constructs as previously described (18Brummelkamp T.R. Kortlever R.M. Lingbeek M. Trettel F. MacDonald M.E. van Lohuizen M. Bernards R. J. Biol. Chem. 2002; 277: 6567-6572Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Following identification of Rem2 from the functional screen, wild type mouse Rem2 was constructed by PCR from the same library used in the screen (whole brain pLib-cDNA retroviral library from Clontech). Primers for Rem2 with a N-terminal HA tag where designed and cloned back into pLib vector (same as cDNA library) using EcoRI and NotI sites (forward, 5′-gatcGAATTCGCCATGGCTTACCCATACGATGTTCCAGATTACGCGCACGTGCCCTACAAACACGAGCTG-3′, and reverse, 5′-gatcGCGGCCGCTCAGAGCACAGAGAGGTCGTGACATGACCTGGA-3′). We reasoned that wild type (WT) Rem2 was more biologically relevant than a constitutively active mutant because WT Rem2 would be dependent on GDP-GTP cycling and specific GEFs or GAPs in the cell. 4Dr. John Collard, personal communication. For transient expression studies, wild type Rem2 cloned into Myc-tagged pME18S constructs were a kind gift from Walter Hunziker and Pascal Beguin (13Beguin P. Mahalakshmi R.N. Nagashima K. Cher D.H. Kuwamura N. Yamada Y. Seino Y. Hunziker W. Biochem. J. 2005; 390: 67-75Crossref PubMed Scopus (62) Google Scholar, 15Beguin P. Mahalakshmi R.N. Nagashima K. Cher D.H. Takahashi A. Yamada Y. Seino Y. Hunziker W. J. Cell Sci. 2005; 118: 1923-1934Crossref PubMed Scopus (69) Google Scholar). Rem2 14-3-3 and CaM point binding mutants (Myc tagged) were also provided by Walter Hunziker and Pascal Beguin. For mouse p53 shRNAi the sequence is published elsewhere (19Dirac A.M. Bernards R. J. Biol. Chem. 2003; 278: 11731-11734Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). For mouse p19ARF we targeted the following sequence: GTTCGTGCGATCCCGGAGA. RNAi directed against 14-3-3γ was cloned into pSUPER and was a gift from Dr. T. Suzuki (20Sumioka A. Nagaishi S. Yoshida T. Lin A. Miura M. Suzuki T. J. Biol. Chem. 2005; 280: 42364-42374Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). For mouse Rem2 we targeted the following Rem2 sequences: 1) GCGGCGGGCCCAAGCTGTA, 2) GCCCGCTCCCGGGAGGTAT, and 3) CGGGGGATGCCTTTCTCAT. Western Blotting, Immunohistochemistry, and Fluorescence-activated Cell Sorter–Western blots were probed with antibodies against: 14-3-3γ (Santa Cruz Biotechnology, Santa Cruz, CA), α-tubulin (Sigma), p53 (Santa Cruz), p21 (Santa Cruz), p14ARF (Neomarker), p19ARF (Abcam), CDK4 (Santa Cruz), HA tag (Santa Cruz), Myc tag (4AE Roche), Cyclin A (Santa Cruz), Cyclin D (Santa Cruz), p107 (Santa Cruz), and p27 (Santa Cruz). A polyclonal antibody for Rem2 was developed against the following peptide sequence CVPRNAKFFKQRSRS. The day 28 bleed was used at 1:750 dilution in 1% milk TBS, Tween 20. Western blots were developed using enhanced chemiluminescence (Amersham Biosciences) following the instructions of the manufacturer. For visualization of actin, we used BODIPY 650/665-phalloidin (Molecular Probes, Leiden, Netherlands). For detection of trimethylated histone H3 at lysine 9 a polyclonal anti-H3K9-Me antibody (Cell Signaling Technology) and fluorescein isothiocyanate-conjugated goat anti-rabbit IgG were used. 300 cells displaying only high levels of signal were selected and the number of cells containing trimethylated histone H3 lysine 9 were counted by light microscopy (equivalent expression levels to cells at 39 °C). Myc-tagged Rem2 was detected using 9E10 and Alexa 488-conjugated goat anti-mouse IgG (Molecular Probes) as a secondary antibody. Nuclei were stained using TO-PRO-3 (Molecular Probes). Cells were embedded in Vectashield mounting medium (Vector Laboratories, Burlington, VT) and analyzed by confocal microscopy using a Zeiss LSM510 with the appropriate filter settings. For the quantification of nuclear exclusion of Rem2 and its mutants, 60-70 Rem2 expressing cells from three independent experiments were randomly selected and a single confocal xy-plane was imaged through the middle of the cell. Rem2 exclusion was arbitrarily scored as none, partial, or complete based on its colocalization with the nuclear TO-PRO-3. Reverse Transcriptase-PCR–Total RNA was isolated using TRIzol reagent (Invitrogen) and a subsequent clean-up RNeasy protocol (Qiagen Inc., Valencia, CA). 250 ng of total RNA was reverse transcribed into cDNA following instructions from the manufacturers and the resulting cDNA was analyzed by PCR. Primers for p19ARF used were: Exon 1β forward: 5′-GTCGCAGGTTCTTGGTCACTGTGA-3 and Exon 2 of p16 reverse: 5′-GTCCTCGCAGTTCGAATCTGC-3′. Primers for mouse Rem2 were: forward, 5′-CGTGGGGGAGAGTGGCGTGGG-3′ and reverse, 5′-ACGAGTGTTGTGGTGGAGAGC-3′ producing a 445-bp fragment. Primers were annealed at 60 °C at 27-35 cycles as described in the figures. Colony Formation Assay and β-Galactosidase Staining–Cells were plated at low density after retroviral infection with genes of interest and left at 32 °C overnight. The following day, plates were shifted to 39 °C and left for 2-6 weeks, depending on the gene being investigated. Plates were then washed, fixed in methanol, and stained with Coomassie Blue for 1 h, washed, and air-dried. β-Galactosidase staining was performed as described before and 300 cells counted for blue signal per condition described (21Dimri G.P. Lee X. Basile G. Acosta M. Scott G. Roskelley C. Medrano E.E. Linskens M. Rubelj I. Pereira-Smith O. Peacocke M. Campisi J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9363-9367Crossref PubMed Scopus (5650) Google Scholar). Statistical analysis of cell numbers of colony formation assay was performed using Student's t test with Excel software. Luciferase Assays–Equal numbers of HEK cells were transfected with 1-3 mg of CMV-RGK, 2 mg of luciferase construct, and 1 mg of CMV-β-galactosidase. Total protein was measured and 4 mg measured for luciferase activity and 8 mg for β-galactosidase activity. Luciferase counts were corrected to β-galactosidase levels. Matrigel Tube Formation, Scratch Assay, and RIP1 tag2 Model–Matrigel tube formation assay was performed by applying growth factor-reduced Matrigel in 24-well plates, allowed to polymerize, and 1 × 105 cells were seeded in serum-free media for 16 h. Tube formation was photographed and assessed. Scratch assay was performed by plating an equal number of cells in gelatin-coated plates, grown to confluence, scratched at least five times with a plastic pipette tip, and migrated into the wound photographed the following day. Frozen sections of the angiogenic model of pancreatic carcinoma, RIP1 tag2, were provided by Dr. Oriol Casanovas. Briefly, sections were fixed in acetone at -20 °C for 10 min, blocked in 0.3% H2O2 for 5 min, 20% horse serum for 30 min and 1:200 dilution of Rem2, rabbit serum (from control rabbit), or CD31 (Pharmingen) applied overnight in 10% horse serum/phosphate-buffered saline. The next day sections were developed using an ABC method (Vector) with dimethylaminoazobenzene (rapid stain), counterstained with hematoxylin (1:5 dilution), mounted in DepEx, and visualized with light microscopy. Blood vessel count was assessed in 10-μm serial sections (first section for CD31, second section for Rem2) using “hot spot” methodology described previously (1Weidner N. Folkman J. Important Adv. Oncol. 1996; : 167-190PubMed Google Scholar). Functional Screen in Endothelial Cells Identifies Rem2–To study the genes involved in the p53/Rb pathway in mouse endothelial cells, we subcloned a mouse endothelial cell line expressing a temperature-sensitive mutant of SV40 T antigen (ts T) to identify a clone with no background in repetitive experiments (16Kanda S. Landgren E. Ljungstrom M. Claesson-Welsh L. Cell Growth & Differ. 1996; 7: 383-395PubMed Google Scholar). These cells were given the acronym LTEC5 for Large T endothelial cell, clone number 5. This mutant binds and inactivates both pRb and p53 at 32 °C allowing the cells to proliferate indefinitely at this temperature. At 39 °C, however, the mutant T antigen is degraded, so that p53 and pRb are released, resulting in cell cycle arrest (see Fig. 1A, inset i). Acidic β-galactosidase staining of endothelial cells and their flattened morphology suggests that 91% of cells undergo a senescence-like arrest at 39 °C (Fig. 1A, insets i and ii). To further characterize the cell cycle arrest we assessed the state of trimethylation of histone H3 lysine 9, an established marker of cell senescence (22Braig M. Lee S. Loddenkemper C. Rudolph C. Peters A.H. Schlegelberger B. Stein H. Dorken B. Jenuwein T. Schmitt C.A. Nature. 2005; 436: 660-665Crossref PubMed Scopus (940) Google Scholar). We found that 99.6% of cells express high levels of trimethylated histone H3 lysine 9 at 39 °C, and only 1.3% of cells at 32 °C express similar levels to the non-permissive temperature (Fig. 1A, insets iii and iv). Furthermore, we found that the endothelial cell cycle arrest is reversible supporting other data that rodent senescence is p53 dependent and reversible (see supplementary information, Fig. S1, panel A) (19Dirac A.M. Bernards R. J. Biol. Chem. 2003; 278: 11731-11734Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). Finally, fluorescence-activated cell sorter analysis of the mouse endothelial cells demonstrates that they arrest in the G1 phase of the cell cycle (see supplementary information, Fig. S1, panel B). To investigate whether cell cycle arrest in these endothelial cells is dependent on the p53 pathway, a main regulator of the G1 phase of the cell cycle, we used shRNA-mediated persistent RNA interference to knock down the p53 and p19ARF tumor suppressor genes. Fig. 1B shows that reduced levels of p53 and p19ARF proteins rescue the temperature shift-induced cell cycle arrest and thus causes immortalization at 39 °C. The results indicate that the endothelial cell arrest at 39 °C is dependent on the p53 pathway in endothelial cells (Fig. 1B). Given that loss of p19ARF or p53 resulted in rescue of the cell cycle arrest, we propose an in vitro genetic model to screen for genes that can modulate the p53-induced senescence-like cell cycle arrest in mouse endothelial cells (Fig. 1, A and B). A functional genomic screen was performed in the ts T mouse endothelial cells using a mouse whole brain cDNA library (Clontech). Sequencing genomic DNA recovered from a rare colony that proliferated at 39 °C identified Rem2. Rem2 is a new member of the RGK family of Ras-like GTPases (Rad, Gem (Kir), Rem and Rem2), which have a previously described role in aspects of the angiogenic cascade (12Piddini E. Schmid J.A. de Martin R. Dotti C.G. EMBO J. 2001; 20: 4076-4087Crossref PubMed Scopus (66) Google Scholar, 14Pan J.Y. Fieles W.E. White A.M. Egerton M.M. Silberstein D.S. J. Cell Biol. 2000; 149: 1107-1116Crossref PubMed Scopus (38) Google Scholar). Consequently, we focused on Rem2. Re-cloning wild type Rem2 into the same vector from the library, we found that ectopic Rem2 rescued the p53 cell cycle arrest to immortalize endothelial cells (Fig. 2A). As a control already known to regulate the p53 pathway via p19ARF suppression (18Brummelkamp T.R. Kortlever R.M. Lingbeek M. Trettel F. MacDonald M.E. van Lohuizen M. Bernards R. J. Biol. Chem. 2002; 277: 6567-6572Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar), we overexpressed TBX3 and found that the endothelial cell cycle arrest was rescued (Fig. 2, A and B). Oncogenic Ras (Ras v12) expression caused endothelial cell cycle arrest as expected (Fig. 2, A and B). Analysis of expression of the ectopic Rem2 protein after 2 weeks reveals that it is not degraded at 39 °C and therefore was sufficient to rescue a p53 cell cycle arrest (Fig. 2C). Therefore, we focused on the role of Rem2 in endothelial cells. Rem2 Suppresses p19ARF/p14ARF Expression in Endothelial Cells–To elucidate the mechanism of the Rem2-induced bypass of the p53-dependent endothelial cell cycle arrest, endothelial cell clones that overexpress Rem2 at 39 °C were picked and expanded for further analysis. A total of 13 clones were first analyzed for SV40 Large T antigen (T) and Rem2 expression levels (see supplementary information, Fig. S2, panel A). Next we determined the p53 status of those endothelial clones that expressed various levels of Rem2 and not T (see supplementary information, Fig. S2 panel B). Cisplatin causes activation of wild type p53 and subsequently one of its targets, p21CIP, making it a useful tool to determine whether p53 is wild type and functional. Following treatment with cisplatin, p53 and p21CIP were both activated in all endothelial cell clones, demonstrating that ectopic Rem2 expression was able to bypass a wild type p53-induced cell cycle arrest and that this response was uniform for all clones. We continued to investigate those endothelial clones that expressed a range from low to high levels of Rem2 protein expression with a wild type p53. Because p53/Rb regulate the G1 phase of the cell cycle (see supplementary information, Fig. S1, panel B), we next performed Western blot analyses of genes regulating the G1-S phase of the endothelial cell cycle (see supplementary information, Fig. S2, panel C) and found that the most significant change caused by ectopic Rem2 was the suppression of p19ARF protein levels (Fig. 3A). To verify that ectopic Rem2 specifically regulated the p19ARF protein levels, we designed three individual shRNA constructs to knock down Rem2, resulting in ∼70% knockdown (Fig. 3B, inset i). The use of three shRNAi constructs reduces the chances of off target effects to nil. Loss of ectopic Rem2 by shRNAi resulted in restoration of the p53 pathway, with increased p19ARF and p53 protein levels following Rem2 RNAi treatment with any of the three individual constructs (Fig. 3B, inset ii). Moreover, functional analyses demonstrated that loss of ectopic Rem2 resulted in reduced colony formation at 39 °C, not seen in p53 and Rem2 RNAi double-treated cells (Fig. 3B, inset iii, first 4 panels). This data supports the idea that ectopic Rem2 suppresses p19ARF expression leading to increased proliferation. Loss of endogenous Rem2 in p53 null-treated endothelial cells had no effect on colony formation assay suggesting that activation of Rem2 is needed before suppressing p19ARF expression (Fig. 3B, inset iii, fourth panel). To further establish that the Rem2-mediated effects on p19ARF are needed for proliferation we overexpressed p19ARF in Rem2 expressing mouse endothelial cell clones by retroviral infection (70% infection levels) and performed colony formation assay. Re-introduction of p19ARF expression inhibited Rem2-induced proliferation demonstrating that p19ARF is essential for the proliferative effects of ectopic expression of Rem2 (Fig. 3B, inset iii, bottom left panel). The use of a GFP RNAi construct demonstrates that the effects of the Rem2 RNAi on proliferation are specific (Fig. 3B, inset iii, bottom right panel). Quantification of the colony formation assay demonstrates the clear differences between treatments (Fig. 3B, inset iv). To further investigate the mechanism of regulation of p19ARF by Rem2, we assessed the mRNA levels following Rem2 ectopic expression. Using semiquantitative reverse transcriptase-PCR we found that the endogenous levels of Rem2 mRNA are low in mouse brain capillary endothelial cells (Fig. 3C, last lane). We found that in mouse endothelial cell clones, which at 39 °C expressed low to moderate levels of Rem2 based on protein expression (see supplementary information, Fig. S1, panel B), p19ARF was transcriptionally suppressed (Fig. 3C). Next, we determined where ectopic Rem2 localizes in the cells. In agreement with previous work we found that ectopic Rem2 was localized in the cytoplasm of mouse endothelial Rem2 clones (13Beguin P. Mahalakshmi R.N. Nagashima K. Cher D.H. Kuwamura N. Yamada Y. Seino Y. Hunziker W. Biochem. J. 2005; 390: 67-75Crossref PubMed Scopus (62) Google Scholar) (Fig. 3D and see supplementary information, Fig. S4, panel B). The use of antibodies against the HA tag confirm the cytoplasmic location of the ectopic Rem2 protein. This suggests that the effect of Rem2 on the p53 pathway is indirect. To functionally assess if Rem2 can accelerate primary HUVEC proliferation rates and to rule out any effects of SV40 large T antigen that may have been undetected in the mouse endothelial cells (see supplemental Fig. 2B), we overexpressed wild type Rem2 and measured primary HUVEC proliferation. Ectopic WT Rem2 increased HUVEC proliferation rates above controls, demonstrating that ectopic Rem2 induces endothelial cell proliferation (Fig. 3E, inset i). Importantly we found that ectopic Rem2 completely suppressed p14ARF protein expression in HUVEC, supporting the data in mouse endothelial cells that overexpressed Rem2 suppresses p14ARF and p19ARF expression (Fig. 2E, inset ii). 14-3-33 Protein Binding Is Involved in Cell Localization of Rem2 and Suppression of p19ARF Expression–We found Rem2 in the cytoplasm and so investigated a role for 14-3-3 proteins in mediating the Rem2 effects for three reasons. First, Rem2 and 14-3-3 proteins have been shown to physically interact in the cytoplasm (13Beguin P. Mahalakshmi R.N. Nagashima K. Cher D.H. Kuwamura N. Yamada Y. Seino Y. Hunziker W. Biochem. J. 2005; 390: 67-75Crossref PubMed Scopus (62) Google Scholar, 15Beguin P. Mahalakshmi R.N. Nagashima K. Cher D.H. Takahashi A. Yamada Y. Seino Y. Hunziker W. J. Cell Sci. 2005; 118: 1923-1934Crossref PubMed Scopus (69) Google Scholar). Second, 14-3-3 proteins have been found to be associated with actively expressed gene promo
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