Up-regulation of Nucleolin mRNA and Protein in Peripheral Blood Mononuclear Cells by Extracellular-regulated Kinase
2001; Elsevier BV; Volume: 276; Issue: 2 Linguagem: Inglês
10.1074/jbc.m009435200
ISSN1083-351X
AutoresCara J. Westmark, James S. Malter,
Tópico(s)Genomics and Chromatin Dynamics
ResumoThe signal transduction pathways regulating nucleolin mRNA and protein production have yet to be elucidated. Peripheral blood mononuclear cells treated with phorbol 12-myristate 13-acetate showed steady state levels of nucleolin mRNA that were 2–2.5-fold greater than untreated control cells. The up-regulation of nucleolin mRNA was substantially repressed by U0126, a specific inhibitor that blocks phosphorylation of extracellular-regulated kinase (ERK). Calcium ionophores A23187 and ionomycin also activated ERK and substantially elevated nucleolin mRNA levels, demonstrating phorbol 12-myristate 13-acetate and calcium signaling converge on ERK. Drugs that affected protein kinase C, protein kinase A, and phospholipase C signal transduction pathways did not alter nucleolin mRNA levels significantly. The half-life of nucleolin mRNA increased from 1.8 h in resting cells to 3.2 h with phorbol ester activation, suggesting ERK-mediated posttranscriptional regulation. Concomitantly, full-length nucleolin protein was increased. The higher levels of nucleolin protein were accompanied by increased binding of a 70-kDa nucleolin fragment to the 29-base instability element in the 3′-untranslated region of amyloid precursor protein (APP) mRNA in gel mobility shift assays. Supplementation of rabbit reticulocyte lysate with nucleolin decreased APP mRNA stability and protein production. These data suggest ERK up-regulates nucleolin posttranscriptionally thereby controlling APP production. The signal transduction pathways regulating nucleolin mRNA and protein production have yet to be elucidated. Peripheral blood mononuclear cells treated with phorbol 12-myristate 13-acetate showed steady state levels of nucleolin mRNA that were 2–2.5-fold greater than untreated control cells. The up-regulation of nucleolin mRNA was substantially repressed by U0126, a specific inhibitor that blocks phosphorylation of extracellular-regulated kinase (ERK). Calcium ionophores A23187 and ionomycin also activated ERK and substantially elevated nucleolin mRNA levels, demonstrating phorbol 12-myristate 13-acetate and calcium signaling converge on ERK. Drugs that affected protein kinase C, protein kinase A, and phospholipase C signal transduction pathways did not alter nucleolin mRNA levels significantly. The half-life of nucleolin mRNA increased from 1.8 h in resting cells to 3.2 h with phorbol ester activation, suggesting ERK-mediated posttranscriptional regulation. Concomitantly, full-length nucleolin protein was increased. The higher levels of nucleolin protein were accompanied by increased binding of a 70-kDa nucleolin fragment to the 29-base instability element in the 3′-untranslated region of amyloid precursor protein (APP) mRNA in gel mobility shift assays. Supplementation of rabbit reticulocyte lysate with nucleolin decreased APP mRNA stability and protein production. These data suggest ERK up-regulates nucleolin posttranscriptionally thereby controlling APP production. amyloid precursor protein casein kinase 2 tricyclodecan-9-yl xanthogenate 5,6-dichlorobenzimidazole riboside enhanced chemiluminescence extracellular-regulated kinase 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphorylcholine heterogeneous nuclear ribonucleoprotein mitogen-activated protein kinase 1-oleoyl-2-acetyl-sn-glycerol (C18:1,[cis]-9/C2:0) peripheral blood mononuclear cells phosphate-buffered saline protein kinase A protein kinase C phospholipase C phorbol 12-myristate 13-acetate rabbit reticulocyte lysate N-(6-phenylhexyl)-5-chloro-1-naphthalenesulfonamide bis[2-aminophenylthio]butadiene untranslated region Alzheimer's disease is characterized by the presence of senile plaques and neurofibrillary tangles in brain tissue. The major proteinaceous material in the senile plaques is β-amyloid, a 40–42-amino acid peptide derived from the amyloid precursor protein (APP).1 Investigation of the molecular regulation of APP mRNA and protein levels is vital to understanding β-amyloid accumulation and deposition in Alzheimer's disease. Our laboratory has previously demonstrated that two RNA-binding proteins, nucleolin and heterogeneous nuclear ribonucleoprotein C (hnRNP C), bind to a 29-base instability element in the 3′-untranslated region (UTR) of APP mRNA (1Zaidi S.H. Malter J.S. J. Biol. Chem. 1995; 270: 17292-17298Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). In rabbit reticulocyte lysate (RRL), hnRNP C binding to the 29-base element stabilized APP mRNA resulting in a 6-fold increase in APP protein production (2Rajagopalan L.E. Westmark C.J. Jarzembowski J.A. Malter J.S. Nucleic Acids Res. 1998; 26: 3418-3423Crossref PubMed Scopus (90) Google Scholar). The role of nucleolin was not determined in these experiments. Nucleolin (C23) is a 110-kDa multifunctional phosphoprotein. It is an abundant nucleolar protein (3Lapeyre B. Bourbon H. Amalric F. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 1472-1476Crossref PubMed Scopus (313) Google Scholar) found in the fibrillar centers and on organizer regions of metaphase chromosomes (4Lischwe M.A. Richards R.L. Busch R.K. Busch H. Exp. Cell Res. 1981; 136: 101-109Crossref PubMed Scopus (130) Google Scholar). Nucleolin plays a role in chromatin decondensation (5Erard M.S. Belenguer P. Caizergues-Ferrer M. Pantaloni A. Amalric F. Eur. J. Biochem. 1988; 175: 525-530Crossref PubMed Scopus (177) Google Scholar), the transcription and processing of rRNA (6Egyhazi E. Pigon A. Chang J.H. Ghaffari S.H. Dreesen T.D. Wellman S.E. Case S.T. Olson M.O. Exp. Cell Res. 1988; 178: 264-272Crossref PubMed Scopus (42) Google Scholar, 7Jordan G. 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Jucker M. Kleinman H.K. J. Neurosci. Res. 1995; 42: 314-322Crossref PubMed Scopus (60) Google Scholar), growth factors (19Take M. Tsutsui J. Obama H. Ozawa M. Nakayama T. Maruyama I. Arima T. Muramatsu T. J. Biochem. (Tokyo). 1994; 116: 1063-1068Crossref PubMed Scopus (103) Google Scholar), and the complement inhibitor, factor J (20Larrucea S. Gonzalez-Rubio C. Cambronero R. Ballou B. Bonay P. Lopez-Granados E. Bouvet P. Fontan G. Fresno M. Lopez-Trascasa M. J. Biol. Chem. 1998; 273: 31718-31725Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Central to nucleolin functions are RNA/DNA binding and helicase activities (21Tuteja N. Huang N.W. Skopac D. Tuteja R. Hrvatic S. Zhang J. Pongor S. Joseph G. Faucher C. Amalric F. Falaschi A. Gene (Amst.). 1995; 160: 143-148Crossref PubMed Scopus (87) Google Scholar). The cDNA for nucleolin has been cloned and codes for a 707-amino acid protein with at least three functional domains (3Lapeyre B. Bourbon H. Amalric F. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 1472-1476Crossref PubMed Scopus (313) Google Scholar, 22Srivastava M. Fleming P.J. Pollard H.B. Burns A.L. FEBS Lett. 1989; 250: 99-105Crossref PubMed Scopus (114) Google Scholar). The 5′-flanking region and the first intron contain a high GC content similar to the housekeeping genes. The 5′ promoter has one atypical TATA box (GTTA), one CCAAT box, three reverse complements of CCAAT (ATTGG), two pyrimidine-rich stretches, and numerous potential transcription factor-binding sites, whereas the 3′-UTR has five homology blocks in a 100-base region (23Srivastava M. McBride O.W. Fleming P.J. Pollard H.B. Burns A.L. J. Biol. Chem. 1990; 265: 14922-14931Abstract Full Text PDF PubMed Google Scholar). There are several distinct features in the amino acid sequence of nucleolin that enable this extraordinary protein to display such a vast array of functions (3Lapeyre B. Bourbon H. Amalric F. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 1472-1476Crossref PubMed Scopus (313) Google Scholar, 7Jordan G. Nature. 1987; 329: 489-490Crossref PubMed Scopus (179) Google Scholar). At the amino-terminal end of the molecule, there are six (G/A/V)TP(G/A/V)KK(G/A/V)(G/A/V) repeats followed by several glutamic/aspartic acid stretches separated by basic sequences and four potential serine phosphorylation sites. The central region, a putative globular domain, contains alternating hydrophobic and hydrophilic stretches. There are four 90-residue repeats, each containing an RNP-like consensus sequence (24Bugler B. Bourbon H. Lapeyre B. Wallace M.O. Chang J.H. Amalric F. Olson M.O. J. Biol. Chem. 1987; 262: 10922-10925Abstract Full Text PDF PubMed Google Scholar). The carboxyl terminus is glycine/arginine-rich with regularly spaced phenylalanine and dimethylarginine residues (25Lapeyre B. Amalric F. Ghaffari S.H. Rao S.V. Dumbar T.S. Olson M.O. J. Biol. Chem. 1986; 261: 9167-9173Abstract Full Text PDF PubMed Google Scholar). The central 40-kDa domain of nucleolin containing the four RNA recognition motifs is responsible for the specificity of RNA binding, and the carboxyl-terminal domain enhances interaction but does not contribute to ligand specificity (26Ghisolfi L. Kharrat A. Joseph G. Amalric F. Erard M. Eur. J. Biochem. 1992; 209: 541-548Crossref PubMed Scopus (101) Google Scholar). The carboxyl-terminal domain contains an ATP-dependent duplex-unwinding activity, and phosphorylation enhances this helicase activity (21Tuteja N. Huang N.W. Skopac D. Tuteja R. Hrvatic S. Zhang J. Pongor S. Joseph G. Faucher C. Amalric F. Falaschi A. Gene (Amst.). 1995; 160: 143-148Crossref PubMed Scopus (87) Google Scholar, 27Ghisolfi L. Joseph G. Amalric F. Erard M. J. Biol. Chem. 1992; 267: 2955-2959Abstract Full Text PDF PubMed Google Scholar). We were initially interested in determining how nucleolin regulation might affect APP mRNA levels and stability. Preliminary studies suggested cytokine-mediated signaling through protein kinases altered nucleolin mRNA levels in PBMC. We found that activation of the extracellular-regulated kinase (ERK)-specific mitogen-activated protein kinase (MAPK) pathway significantly up-regulated nucleolin mRNA levels independently of protein kinase C (PKC). Phorbol 12-myristate 13-acetate (PMA) treatment also resulted in higher levels of full-length nucleolin protein and the disappearance of the 47-kDa nucleolin cleavage fragment. In gel mobility shift assays, lysates from phorbol ester and ionomycin-treated peripheral blood mononuclear cells (PBMC) contained nucleolin that bound to the 29-base instability element of APP mRNA. In RRL supplemented with nucleolin protein, APP mRNA decayed with a shortened half-life of 105 min but was indefinitely stable in RRL supplemented with globin. The loss of APP mRNA stability resulted in decreased APP production. Therefore, ERK activation stabilizes nucleolin mRNA resulting in increased nucleolin levels and accelerated decay of APP mRNA. Protease inhibitor mixture (catalog number P2714), Escherichia coli tRNA, RNase TI, 4α-phorbol, PMA, 1-oleoyl-2-acetyl-sn-glycerol (C18:1,[cis]-9/C2:0) (OAG), calcium ionophore A23187, ionomycin calcium salt, thapsigargin, dantrolene, forskolin, 1,9-dideoxyforskolin, cAMP, (Rp)-cAMPS, imipramine, wortmannin, 17β-estradiol, corticosterone, and 5,6-dichlorobenzimidazole riboside (DRB) were from Sigma.N-(6-Phenylhexyl)-5-chloro-1-naphthalenesulfonamide (SC-9), tricyclodecan-9-yl xanthogenate (D609), and 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphorylcholine (Et-18-OCH3) were from Calbiochem. Klenow enzyme, RNasin, bis[2-aminophenylthio]butadiene (U0126), and RRL were from Promega (Madison, WI). Lymphoprep density gradient medium and RPMI 1640 were from Life Technologies, Inc., and TRI-reagent was purchased from Molecular Research Center, Inc. (Cincinnati, OH). The enhanced chemiluminescence (ECL) Western blotting detection kit, [α-32P]dCTP andl-[35S]methionine were from Amersham Pharmacia Biotech; the nylon transfer membrane was from Fisher, and the QuikHyb hybridization solution and NucTrap probe purification columns were supplied by Stratagene (La Jolla, CA). The T7 and SP6 mMessage mMachine in vitro transcription kits and oligo(dT)-cellulose were purchased from Ambion, Inc. (Austin, TX). Peripheral blood was collected by phlebotomy from consenting, healthy laboratory personnel and was anticoagulated with heparin. The experimental protocol was approved by the University of Wisconsin Hospital Human Subjects Review Committee, which meets National Institutes of Health guidelines. PBMC were isolated by Ficoll-Paque density centrifugation, stimulated with various kinase effectors, and lysed in Tri-reagent as described previously. 2C. J. Westmark and J. S. Malter, submitted for publication. RNA was electrophoresed on formaldehyde-agarose gels, transferred to nylon membranes, and hybridized with radiolabeled cDNA probes. The samples were assayed in at least triplicate, normalized to a control RNA (18 S or ribosomal protein S26 mRNA), and plotted as a percentage of total nucleolin mRNA. Error bars depict the standard error of the mean, and p values were calculated by the Student's t test. Cytoplasmic lysates were prepared as described previously (29Fukuda H. Nishida A. Saito H. Shimizu M. Yamawaki S. Neurochem. Int. 1994; 25: 567-571Crossref PubMed Scopus (26) Google Scholar). Briefly, cultured PBMC (2 ml at 5 × 106 cells/ml) were scraped from tissue culture wells, spun at 2,000 × g for 30 s in a Stratagene picofuge microcentrifuge, washed three times with ice-cold phosphate-buffered saline (PBS), and resuspended in 50 μl of ice-cold buffer containing 25 mm Tris, pH 8, 0.1 mmEDTA, and 1× protease inhibitor mixture. The resuspended cells were lysed by five freeze (−80 °C)/thaw (37 °C) cycles and spun at 15,000 × g for 15 min at 4 °C. The supernatants (cytoplasmic lysates) were transferred to fresh tubes and frozen at −80 °C. The protein concentration of the lysates was quantitatively determined with Bio-Rad protein assay dye reagent per the manufacturer's instructions. Lysate (10 μg) in a 12-μl volume was mixed with 4 μl (4×) SDS reducing buffer (62.5 mm Tris-HCl, pH 6.8, 10% glycerol, 2% SDS, 5% β-mercaptoethanol, 0.06% bromphenol blue), boiled for 5 min, and analyzed on a 12% SDS-polyacrylamide gel. The gel was equilibrated in Bjerrum and Schafer-Nielsen transfer buffer (48 mm Tris, 39 mm glycine, 20% methanol, 1.3 mm SDS, pH 9.2), and the proteins were transferred to 0.2-μm pure nitrocellulose membrane by semi-dry electrophoretic transfer on a Bio-Rad Trans-Blot SD apparatus at 15 V for 45 min. The nitrocellulose membrane was blocked for 1 h in 5% nonfat dry milk and stained by ECL per the manufacturer's directions. The primary antibody was anti-nucleolin (1:1000) (kindly provided by Dr. Raymond Petryshyn, Children's National Medical Center, Washington, D. C.) and the secondary antibody was anti-rabbit horseradish peroxidase (1:2000). The membrane was exposed to x-ray film for 1 min. APP106 template was prepared by amplification of nucleotides 2415–2520 of the plasmid pT7APP751wtΔHindIIIT90 (2Rajagopalan L.E. Westmark C.J. Jarzembowski J.A. Malter J.S. Nucleic Acids Res. 1998; 26: 3418-3423Crossref PubMed Scopus (90) Google Scholar) with the primers 5′-CACAATACGACTCACTATAGGGAACTTGAATTAATCCACA-3′ (APP cDNA(2415–2432)) and 5′-ACAGCTAAATTCTTTACAGT-3′ (APP cDNA(2520–2501)) by PCR (1 min at 94 °C, 1 min at 50 °C, and 10 s at 72 °C for 35 cycles). The 5′ primer included a T7 RNA polymerase promoter sequence, which is underlined. The PCR product was extracted with Tris-saturated phenol/chloroform and precipitated with 0.1 volume of sodium acetate and 2 volumes of ethanol before gel purification on an 8% nondenaturing polyacrylamide gel. Radiolabeled RNA probes were prepared according to Promega's standard transcription protocol for T7 RNA polymerase with each labeling reaction containing 200 ng of APP106 template and 50 μCi of [α-32P]UTP. Reactions were incubated for 60 min at 37 °C and stopped by the addition of 2 units of RNase-free DNase I for 15 min at 37 °C. Samples were extracted with water-saturated phenol/chloroform, and unincorporated isotope was removed by passage through NucTrap probe purification columns. The binding reactions were performed similarly to the procedures used in Ref. 28Malter J.S. Science. 1989; 246: 664-666Crossref PubMed Scopus (371) Google Scholar. Briefly, 2 μg of cytoplasmic lysates were incubated with 1 × 105cpm of APP106 RNA probe in 10% glycerol, 15 mm HEPES, pH 8, 10 mm KCl, 1.0 mm dithiothreitol, 200 ng/μl E. coli tRNA, and 1 unit/μl RNasin in a total volume of 10 μl for 10 min at 30 °C. RNase TI (20 units in 1-μl volume) was added, and samples were digested for 30 min at 37 °C. Reactions were cross-linked on automatic for 5 min in a UV Stratalinker 2400 from Stratagene (La Jolla, CA) on ice prior to the addition of 3.5 μl of 4× SDS reducing buffer, heat denaturation for 3 min at 100 °C, and analysis on 12% SDS-polyacrylamide gels. The gels were dried and exposed to x-ray film or a PhosphorImager screen. The construction of pT7APP751wtΔHindIIIT90 has been described (2Rajagopalan L.E. Westmark C.J. Jarzembowski J.A. Malter J.S. Nucleic Acids Res. 1998; 26: 3418-3423Crossref PubMed Scopus (90) Google Scholar). The plasmid pSPκβc containing the β-globin gene cloned between an SP6 promoter and a T stretch was provided by Richard Spritz (University of Wisconsin, Madison, WI), and the nucleolin gene in the pMAM plasmid was a gift from Meera Srivastava (Georgetown University School of Medicine, Washington, D. C). The pMAMnucleolin plasmid was digested with XhoI and NheI to liberate the nucleolin gene. The insert ends were blunted with Klenow and ligated with SmaI-digested pT7T90 transcription vector (provided by Jeff Ross, University of Wisconsin, Madison, WI) to generate pT7nuclcodingT90. The 3′-UTR of nucleolin cDNA was amplified from Jurkat genomic DNA with the primers 5′-ACGAAGTTTGAATAGCTTCT-3′ (nucleolin cDNA(2221–2240)) and 5′-GTAGGAAAAAATGGTTTTGT-3′ (nucleolin cDNA(2518–2499)) by PCR (1 min at 94 °C, 1 min at 50 °C, and 20 s at 72 °C for 35 cycles). The ends were blunted with Klenow and ligated with SmaI-digested pUC-18 generating pUCnucl3′-UTR. pUCnucl3′-UTR was digested with EcoRI and XbaI to liberate the nucleolin 3′-UTR sequence, and the ends were blunted with T4 DNA polymerase and ligated with pT7nuclcodingT90 previously digested with EcoRI and blunted with T4 DNA polymerase. The resulting plasmid, pT7nucleolinT90, contained the entire nucleolin coding region, a short spacer, and the complete nucleolin 3′-UTR sequence cloned between a T7 promoter and a 90-base poly(T) stretch. The plasmids pT7APP751 wtΔHindIIIT90 and pSPκβc were linearized with HindIII, and the plasmid pT7nucleolinT90 was digested with EcoRV. The digests were extracted with phenol and chloroform, precipitated with 0.1 volume ammonium acetate and 2 volumes ethanol, resuspended in water to 0.5 μg/μl, and used as templates (1 μg each) for in vitro transcription with Ambion's T7 and Sp6 mMessage mMachine kits per the manufacturer's directions for 2 h at 37 °C. The capped, polyadenylated mRNA reactions were digested with RNase-free DNase I for 15 min at 37 °C, extracted with water-saturated phenol and chloroform, and purified by oligo(dT)-cellulose chromatography. The oligo(dT)-cellulose was equilibrated in high salt binding buffer containing 0.5m NaCl, 20 mm Tris-Cl, pH 7.5, 1 mmEDTA. The mRNA was heat-denatured for 5 min at 65 °C, chilled in ice, adjusted to high salt binding buffer conditions, and bound to the oligo(dT)-cellulose for 30 min at ambient temperature with mixing. The resin was washed with high salt binding buffer followed by low salt binding buffer (0.1 m NaCl) and finally eluted with TE (pH 7.5) (65 °C). The purity of the mRNA was checked by agarose gel electrophoresis, and the concentration was measured by A260. RRL was programmed with 100 ng of heat-denatured globin versus nucleolin mRNA, and translation proceeded for 3.5 h at 30 °C per the manufacturer's directions. Aliquots of globin- and nucleolin-supplemented RRL were frozen at −80 °C. APP mRNA (50 ng) was incubated with 2 μl of globin- versusnucleolin-supplemented RRL in 10-μl reactions containing 10% glycerol, 15 mm HEPES, pH 8, 10 mm KCl, 1.0 mm dithiothreitol, and 200 ng/μl E. coli tRNA for 10 min at 30 °C. Translation was initiated by the addition of 33 μl of fresh RRL, 0.5 μl of amino acid mixture minus methionine, 0.5 μl of amino acid mixture minus leucine, 1.4 μl of KCl, and 4.6 μl of water. Reactions (50 μl each) were incubated at 30 °C, and 5-μl aliquots were removed at 0, 30, 60, and 120 min. RNA was isolated with Tri-Reagent, and RNA pellets were dissolved in 25 μl of formamide, and 5 μl of each sample was analyzed on formaldehyde-agarose gels. For translation measurements, RRL reactions were similar to the RNA decay reactions althoughl-[35S]methionine was included. Aliquots (2.5 μl each) were removed at 0.5, 1, 2, and 3 h, diluted with 7.5 μl of water, mixed with 3.5 μl of 4× SDS reducing buffer, heated for 20 min at 60 °C, and analyzed on 12% SDS-polyacrylamide gels. The gels were dried and exposed to a PhosphorImager screen. We examined the effect of several drugs that influence MAPK, PKC, protein kinase A (PKA), and phospholipase C (PLC) activity as well as calcium mobilization on the steady state level of nucleolin mRNA in PBMC. To investigate the role of PKC, cells were cultured for 3.25 h in the presence of the phorbol ester PMA. Compared with untreated controls, nucleolin mRNA levels increased by 2.5-fold (Fig.1, lane 2). In all cases, nucleolin mRNA was a single 3.0-kilobase pair transcript. The inactive phorbol ester, 4α-phorbol, did not stimulate nucleolin mRNA accumulation. Two additional activators of PKC, SC-9 and OAG, that stimulate calcium/phospholipid-dependent PKC were also tested (Fig. 1, lanes 3–4). SC-9 had no effect, whereas OAG, a diacylglycerol analog, had a small stimulatory effect (29%) on nucleolin mRNA levels. The PKC inhibitors bisindolylmaleimide I and staurosporine did not block the PMA-mediated increase in nucleolin mRNA levels indicating that PMA acts independently of PKC (data not shown). The effect of several calcium mobilization drugs on nucleolin mRNA levels was next assessed. The ionophores A23187 and ionomycin, which transport calcium from the medium into cells, as well as thapsigargin, which causes the release of calcium from intracellular stores, all increased nucleolin mRNA levels 1.5–2.5-fold above unstimulated controls (Fig. 1, lane 6–8). Dantrolene (lane 9), which blocks intracellular calcium release, had no effect on nucleolin mRNA levels. Therefore, nucleolin mRNA levels were up-regulated in response to drugs that increase intracellular calcium concentrations via release from intracellular stores or import from the extracellular environment. We examined the influence of several activators and inhibitors of PKA and PLC signal transduction on the steady state level of nucleolin mRNA in PBMC. Cyclic AMP, a known activator of PKA, and (Rp)-cAMPS, an inhibitor of PKA, did not change the steady state level of nucleolin mRNA (Fig.2, lanes 4–5). Forskolin activates adenylate cyclase resulting in increased cAMP levels. 1,9-Dideoxyforskolin, a negative control for forskolin, is unable to stimulate adenylate cyclase. Activation of the adenylate cyclase pathway with forskolin did not influence the steady state level of nucleolin mRNA in PBMC (Fig. 2, lane 3). We did observe a 24% increase in nucleolin mRNA levels upon treatment with imipramine, a drug that stimulates PLC in rat brain by a calcium-dependent process (29Fukuda H. Nishida A. Saito H. Shimizu M. Yamawaki S. Neurochem. Int. 1994; 25: 567-571Crossref PubMed Scopus (26) Google Scholar) (Fig.3, lane 4). We did not observe any significant change in nucleolin mRNA levels with D-609, Et-18-OCH3, or wortmannin (Fig. 3, lanes 2, 3and 5), drugs that inhibit phosphatidylcholine-specific PLC, phosphatidylinositol-specific PLC, and phosphatidylinositol 3-kinase, respectively. The steroid hormones, estrogen and corticosterone, also did not affect nucleolin mRNA levels (Fig. 3, lanes 7and 8). Therefore, the imipramine-mediated effects suggest that the PLC pathway partially modulates nucleolin mRNA levels in PBMC.Figure 3PLC pathway agonists do not significantly alter nucleolin mRNA levels in PBMC. Cells were treated 4 h with 20 ng/ml PMA (lane 1), 19 μm D609 (lane 2), 15 μm ET-18-OCH3(lane 3), 100 μm imipramine (lane 4), 10 nm wortmannin (lane 5), 100 nm 17β-estradiol (lane 6), 1.5 μm corticosterone (lane 7), 100 nm17β-estradiol with 1.5 μm corticosterone (est + cort, lane 8), and 0.1% PBS (lane 9) followed by a 15-min chase with DRB. Total RNA (10 μg per lane) was analyzed by Northern blotting. A, representative Northern blot of the nucleolin-specific hybridization signals. B,representative Northern blot of the S26-specific hybridization signals.C, histogram depicting nucleolin mRNA levels normalized to S26 and plotted as a percentage of total nucleolin mRNA. The error bars represent the S.E. for triplicate samples. Student's t test results are as follows: PMA,p = 0.0029; D609, p = 1.0; Et-18-OCH3, p = 0.11; imipramine,p = 0.036; wortmannin, p = 0.17; 17β-estradiol, p = 0.23; corticosterone,p = 0.68; and 17β-estradiol + corticosterone,p = 0.16.View Large Image Figure ViewerDownload Hi-res image Download (PPT) PMA is a known activator of the MAPK signal transduction pathway, so we next assessed the ability of U0126 to block the PMA-mediated increase in nucleolin mRNA levels (Fig. 4). Cells were pretreated with 10 μm U0126 for 15 min prior to the addition of 20 ng/ml PMA for 3 h. The cultures were spiked with 5 μm U0126 after 1 and 2 h with PMA to maintain anti-MEK activity. U0126 blocked the PMA-mediated increase in nucleolin mRNA levels (Fig. 4, lane 3) pointing to the ERK-specific MAPK pathway as a major player in regulating nucleolin mRNA levels. Nucleolin mRNA accumulation could be secondary to increased transcription, decreased degradation, or both. Thus, nucleolin mRNA decay was measured in resting or PMA-treated PBMC. In resting PBMC, the half-life of nucleolin mRNA was 1.8 h (Fig.5), which increased almost 2-fold (t12 = 3.2 h) after treatment for 3.5 h with PMA. Therefore, nucleolin mRNA accumulation after ERK activation can be accounted for by enhanced stability of the message. As mentioned earlier, nucleolin protein has been implicated in many functions including APP mRNA stability. Thus, we examined cell lysates for nucleolin expression by Western blotting and nucleolin binding activity by RNA gel mobility shift assays. There was a time-dependent increase in nucleolin protein levels upon PMA treatment (Fig. 6). On ECL-stained Western blots probed with a polyclonal anti-nucleolin antibody, we observed two prominent nucleolin bands at 47 and 65 kDa in unstimulated PBMC. After PMA stimulation for 15 min, there was an increase in nucleolin fragments of ∼70 kDa, and after 1 h, an increase in the 80-kDa nucleolin fragment. We observed full-length nucleolin protein (100 kDa) after 2 h of PMA treatment which continued to increase for several hours. As full-length nucleolin increased there was a decrease in the 47-kDa nucleolin cleavage product. RNA mobility shifts with radiolabeled APP RNA demonstrated a 2.8-fold increase in nucleolin binding after only 20 min of PMA treatment and a 5.1-fold increase by 135 min (Fig. 7). Our laboratory has previously demonstrated that the 70-kDa nucleolin polypeptide is responsible for the 84-kDa nucleolin/29-base element RNA-protein complex (1Zaidi S.H. Malter J.S. J. Biol. Chem. 1995; 270: 17292-17298Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). In unstimulated cells, there were faint nucleolin-APP RNA complexes in the 60–70-kDa range (Fig. 7, lane 1) which were previously observed only in stimulated cells (16Zaidi S.H. Denman R. Malter J.S. J. Biol. Chem. 1994; 269: 24000-24006Abstract Full Text PDF PubMed Google Scholar). These lower molecular weight complexes are not due to cell stimulation during the PBMC isolation procedure but rather are a consequence of including
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