FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2126451227> ?p ?o ?g. }

• W2126451227 endingPage "15206" @default.
• W2126451227 startingPage "15200" @default.
• W2126451227 abstract "Phorbol 12-myristate 13-acetate (PMA) induces differentiation of human leukemic HL-60 cells into cells with macrophage-like characteristics and enhances the susceptibility of HL-60 cells to the Helicobacter pylori VacA toxin (de Bernard, M., Moschioni., M., Papini, E., Telford, J. L., Rappuoli, R., and Montecucco, C. (1998) FEBS Lett. 436, 218–222). We examined the mechanism by which HL-60 cells acquire sensitivity to VacA, in particular, looking for expression of RPTPβ, a VacA-binding protein postulated to be the VacA receptor (Yahiro, K., Niidome, T., Kimura, M., Hatakeyama, T., Aoyagi, H., Kurazono, H., Imagawa, K., Wada, A., Moss, J., and Hirayama, T. (1999)J. Biol. Chem. 274, 36693–36699). PMA induced expression of RPTPβ mRNA and protein as determined by RNase protection assay and indirect immunofluorescence studies, respectively. Vitamin D3 and interferon-γ, which stimulate differentiation of HL-60 cells into monocyte-like cells, also induced VacA sensitivity and expression of RPTPβ mRNA, whereas 1.2% Me2SO and retinoic acid, which stimulated the maturation of HL-60 into granulocyte-like cells, did not. RPTPβ antisense oligonucleotide inhibited induction of VacA sensitivity and expression of RPTPβ. Double immunostaining studies also indicated that newly expressed RPTPβ colocalized with VacA in PMA-treated HL-60 cells. In agreement with these data, BHK-21 cells, which are insensitive to VacA, when transfected with the RPTPβ cDNA, acquired VacA sensitivity. All data are consistent with the conclusion that acquisition of VacA sensitivity by PMA-treated HL-60 cells results from induction of RPTPβ, a protein that functions as the VacA receptor. Phorbol 12-myristate 13-acetate (PMA) induces differentiation of human leukemic HL-60 cells into cells with macrophage-like characteristics and enhances the susceptibility of HL-60 cells to the Helicobacter pylori VacA toxin (de Bernard, M., Moschioni., M., Papini, E., Telford, J. L., Rappuoli, R., and Montecucco, C. (1998) FEBS Lett. 436, 218–222). We examined the mechanism by which HL-60 cells acquire sensitivity to VacA, in particular, looking for expression of RPTPβ, a VacA-binding protein postulated to be the VacA receptor (Yahiro, K., Niidome, T., Kimura, M., Hatakeyama, T., Aoyagi, H., Kurazono, H., Imagawa, K., Wada, A., Moss, J., and Hirayama, T. (1999)J. Biol. Chem. 274, 36693–36699). PMA induced expression of RPTPβ mRNA and protein as determined by RNase protection assay and indirect immunofluorescence studies, respectively. Vitamin D3 and interferon-γ, which stimulate differentiation of HL-60 cells into monocyte-like cells, also induced VacA sensitivity and expression of RPTPβ mRNA, whereas 1.2% Me2SO and retinoic acid, which stimulated the maturation of HL-60 into granulocyte-like cells, did not. RPTPβ antisense oligonucleotide inhibited induction of VacA sensitivity and expression of RPTPβ. Double immunostaining studies also indicated that newly expressed RPTPβ colocalized with VacA in PMA-treated HL-60 cells. In agreement with these data, BHK-21 cells, which are insensitive to VacA, when transfected with the RPTPβ cDNA, acquired VacA sensitivity. All data are consistent with the conclusion that acquisition of VacA sensitivity by PMA-treated HL-60 cells results from induction of RPTPβ, a protein that functions as the VacA receptor. bovine serum albumin Eagle's minimum essential medium fetal calf serum phorbol 12-myristate 13-acetate interferon phosphate-buffered saline base pair(s) glyceraldehyde-3-phosphate dehydrogenase polymerase chain reaction vitamin D3 Helicobacter pylori is believed to be a major cause of gastritis, gastric ulcer, and gastric cancer (1.Blaser M.J. Trends Microbiol. 1993; 1: 255-260Abstract Full Text PDF PubMed Scopus (186) Google Scholar, 2.Watanabe T. Tada M. Nagai H. Sasaki S. Nakao M. Gastroenterology. 1998; 115: 642-648Abstract Full Text Full Text PDF PubMed Scopus (933) Google Scholar). H. pylori secretes a potent cytotoxin (VacA), which induces cytoplasmic vacuolation in some eukaryotic cells (3.Cover T.L. Blaser M.J. J. Biol. Chem. 1992; 267: 10570-10575Abstract Full Text PDF PubMed Google Scholar, 4.Telford J.L. Ghiara P. Dell'Orco M. Comanducci M. Burroni D. Bugnoli M. Tecce M.F. Censini S. Covacci A. Xiang Z. Papini E. Montecucco C. Parente L. Rappuoli R. J. Exp. Med. 1994; 179: 1653-1658Crossref PubMed Scopus (524) Google Scholar, 5.Yahiro K. Niidome T. Hatakeyama T. Aoyagi H. Kurazono H. Padilla P.I. Wada A. Hirayama T. Biochem. Biophys. Res. Commun. 1997; 238: 629-632Crossref PubMed Scopus (71) Google Scholar, 6.Cover T.L. Trends Microbiol. 1998; 6: 127-128Abstract Full Text Full Text PDF PubMed Google Scholar). Two different VacA genotypes, m1 and m2, have been found in clinical isolates ofH. pylori (1.Blaser M.J. Trends Microbiol. 1993; 1: 255-260Abstract Full Text PDF PubMed Scopus (186) Google Scholar, 6.Cover T.L. Trends Microbiol. 1998; 6: 127-128Abstract Full Text Full Text PDF PubMed Google Scholar, 7.Atherton J.C. Cao P. Peek Jr, R.M. Tummuru M.K. Blaser M.J. Cover T.L. J. Biol. Chem. 1995; 270: 17771-17777Abstract Full Text Full Text PDF PubMed Scopus (1385) Google Scholar, 8.Cover T.L. Mol. Microbiol. 1996; 20: 241-246Crossref PubMed Scopus (259) Google Scholar, 9.Maurizio M. Galli C. de Bernard M. Norais J. Rappuoli R. Montecucco C. Biochem. Biophys. Res. Commun. 1998; 248: 334-340Crossref PubMed Scopus (80) Google Scholar, 10.Dunn B. Cohen H. Blaser M.J. Clin. Microbiol. Rev. 1997; 10: 720-741Crossref PubMed Google Scholar, 11.Kimura M. Goto S. Wada A. Yahiro K. Niidome T. Hatakeyama T. Aoyagi H. Hirayama T. Kondo T. Microb. Pathog. 1999; 26: 45-52Crossref PubMed Scopus (84) Google Scholar). The m2 genotype can be distinguished from the m1 VacA genotype by the presence of a unique middle region locus. The m1 and m2 genotypes differ in cell specificity, suggesting that the two VacA molecules differ in receptor binding (12.Pagliaccia C. de Bernard M. Lupetti P. Ji X. Burroni D. Cover T.L Papini E. Rappuoli R. Telford J.L. Reyrat J.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10212-10217Crossref PubMed Scopus (172) Google Scholar). VacA of the m1 genotype is an oligomeric toxin, composed of 5 or 6 subunits. The 87–95-kDa mature toxin is generated by proteolytic cleavage of a 140-kDa precursor at the bacterial outer membrane (6.Cover T.L. Trends Microbiol. 1998; 6: 127-128Abstract Full Text Full Text PDF PubMed Google Scholar, 13.Manetti R. Massari P. Burroni D. de Bernard M. Marchini A. Olivieri R. Papini E. Montecucco C. Rappuoli R. Telford J.L. Infect. Immun. 1995; 63: 4476-4480Crossref PubMed Google Scholar). The processed VacA caused vacuolation of AZ-521, AGS, and COS-7 cells. Human promyeloblastic HL-60 cells and hamster kidney cells, BHK-21, however, were insensitive to VacA (5.Yahiro K. Niidome T. Hatakeyama T. Aoyagi H. Kurazono H. Padilla P.I. Wada A. Hirayama T. Biochem. Biophys. Res. Commun. 1997; 238: 629-632Crossref PubMed Scopus (71) Google Scholar). de Bernard et al.(19.de Bernard M. Moschioni M. Papini E. Telford J.L. Rappuoli R. Montecucco C. FEBS Lett. 1998; 436: 218-222Crossref PubMed Scopus (14) Google Scholar) reported recently that treatment of HL-60 cells with PMA and butyric acid, agents that stimulate their maturation into macrophage-like and monocyte-like cells, respectively (14.Huberman E. Callaham M. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 1293-1297Crossref PubMed Scopus (490) Google Scholar, 15.Rovera G. Santoli D. Damsky C. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 2779-2783Crossref PubMed Scopus (870) Google Scholar, 16.Koeffler H.P. Golde D.W. Blood. 1980; 56: 344-351Crossref PubMed Google Scholar, 17.Harris P. Ralph P. J. Leukocyte Biol. 1985; 37: 407-422Crossref PubMed Scopus (524) Google Scholar, 18.Collins S. Blood. 1987; 70: 1233-1244Crossref PubMed Google Scholar), also induced VacA sensitivity. We recently reported that VacA is able to interact with target cells by binding to a 250-kDa receptor protein, tyrosine phosphatase (RPTP) β. 1Yahiro, K., Niidome, T., Kimura, M., Hatakeyama, T., Aoyagi, H., Kurazono, H., Imagawa, K., Wada, A., Moss, J., and Hirayama, T. (1999) J. Biol. Chem. 274,36693–36699. To define further whether RPTPβ serves as the VacA receptor, we investigated the relationship between induction of VacA sensitivity and RPTPβ expression in HL-60 cells treated with differentiation-promoting reagents. Agents that promote differentiation of HL-60 cells into macrophage- and monocyte-like cells, but not granulocyte-like cells, enhanced VacA sensitivity by increasing expression of cell surface RPTPβ. Suppression of RPTPβ synthesis with antisense oligonucleotides inhibited receptor expression. Transfection of RPTPβ gene into BHK-21 cells resulted in an induction of VacA sensitivity in these cells. These data support the hypothesis that RPTPβ is the VacA cytotoxin receptor. VacA was purified from the toxin-producing H. pylori strain ATCC49503, according to our published procedure (5.Yahiro K. Niidome T. Hatakeyama T. Aoyagi H. Kurazono H. Padilla P.I. Wada A. Hirayama T. Biochem. Biophys. Res. Commun. 1997; 238: 629-632Crossref PubMed Scopus (71) Google Scholar). In brief, after growth of H. pyloriin Brucella broth containing 0.1% β-cyclodextran at 37 °C for 3–4 days with vigorous shaking in a controlled micro-aerophilic atmosphere of 10% O2 and 10% CO2, VacA was precipitated from culture supernatant using 50% saturated ammonium sulfate and purified by column chromatography on hydroxyapatite, Superose 6HR 10/30, and Resource Q. Purified VacA (200 μg/ml) was stored in TBS buffer (60 mm Tris-HCl buffer, pH 7.7, containing 0.1 m NaCl). VacA concentration was determined by Beads enzyme-linked immunosorbent assay method (21.Nagata H. Wada A. Kurazono H. Yahiro K. Shirasaka D. Ikemura T. Aoyama N. Ping W.A. Makiyama K. Kohno S. Hirayama T. Microb. Pathog. 1999; 26: 103-110Crossref PubMed Scopus (13) Google Scholar). Protein content was measured by the Bradford method using BSA2 as a standard (22.Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217548) Google Scholar). AZ-521 cells (human gastric adenocarcinoma cell line) and BHK-21 (hamster kidney cell line) cells seeded in 96-well culture plates (2 × 104 cells/well, 80 μl) in Dulbecco's modified Eagle's medium and EMEM, respectively, supplemented with 10% FCS and kanamycin (50 μg/ml) were grown as monolayers for 24 h in an atmosphere of 5% CO2 at 37 °C. HL-60 cells were seeded in 96-well tissue culture plates (2 × 104 cells/well, 100 μl) in RPMI 1640 medium supplemented with 10% FCS and kanamycin (50 μg/ml). The cells were incubated with indicated concentrations of PMA or Me2SO for 0, 12, 24, or 48 h. Because PMA was dissolved in 0.1% (v/v) Me2SO, this solution served as a control for PMA. Retinoic acid (100 nm), vitamin D3 (VitD3) (10 nm), or IFN-γ (100 units/ml) (14.Huberman E. Callaham M. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 1293-1297Crossref PubMed Scopus (490) Google Scholar, 23.Collins S.J. Ruscetti F.W. Gallagher R.E. Gallo R.C. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 2458-2462Crossref PubMed Scopus (1428) Google Scholar, 24.Breitman T.R. Selonick S.E. Collins S.J. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 2936-2940Crossref PubMed Scopus (1864) Google Scholar, 25.Tanaka H. Abe E. Miyaura C. Shiina Y. Suda T. Biochem. Biophys. Res. Commun. 1983; 117: 86-92Crossref PubMed Scopus (199) Google Scholar, 26.Ball E.D. Guyre P.M. Shen L. Glynn J.M. Maliszewski C.R. Baker P.E. Fanger M.W. J. Clin. Invest. 1984; 73: 1072-1077Crossref PubMed Scopus (133) Google Scholar, 27.Trayner I.D. Bustorff T. Etches A.E. Mufti G.J. Foss Y. Farzaneh F. Leuk. Res. 1998; 22: 537-547Crossref PubMed Scopus (78) Google Scholar) was added for 24 h. The cells were then incubated with 20 μl of VacA (final concentration, 120 nm) for an additional 8 h at 37 °C. To quantify vacuolation, neutral red uptake into vacuoles was determined as described by Cover et al. (28.Cover T Puryear W. Perez-Perez G.I. Blaser M. Infect. Immun. 1991; 59: 1264-1270Crossref PubMed Google Scholar). Cells were incubated for 5 min at room temperature with 50 μl of freshly prepared 0.05% neutral red in PBS containing 0.3% BSA and then washed three times with 0.1 ml of PBS containing 0.3% BSA. After addition of 0.1 ml of 70% ethanol in water containing 0.4% HCl, absorbance at 540 nm (A 540) was measured. Neutral red uptake was determined by subtracting the A 540 of cells incubated without VacA from that of VacA-treated cells. DNA fragments (6021–6250) from RPTPβ cDNA were generated by PCR amplification and subcloned into pBluescript/KS vector. The identity of the fragments was confirmed by sequencing following established protocols. The plasmid was then linearized by using the XhoI restriction site. Antisense riboprobes were synthesized using the MAXIScript SP6/T7 transcription kit from Ambion. These probes also contained, at their 5′ end, base pairs of pBluescript corresponding to DNA sequences between the T3 promoter and the restriction sites where the DNA fragments were inserted and had a total size of 314 bp. The control probe for GAPDH was synthesized using human GAPDH control template DNA from CLONTECH Laboratories. An amplified GAPDH fragment was generated by PCR using a 3′ primer and transcribed using the T7 promoter sequence. An antisense 184-nucleotide RNA probe was generated using the MAXIScript by Ambion. RNase protection assays were performed according to the protocol contained in the Ribonuclease Protection Assay kit (RPA III; Ambion). Briefly, 20 μg of total RNA from AZ-521 cells and HL-60 cells stimulated by different reagents were used for the RNase A protection assay. Total RNA was hybridized overnight with the RPTPβ and GAPDH probes using hybridization buffer at 42 °C. Afterward, the reaction was treated with RNase, which was then inactivated, and the fragments were precipitated. Protected fragments were detected using 5% sequencing gels followed by Fuji film autoradiography. Sizes of 230 nucleotides for RPTPβ and 184 nucleotides for GAPDH were predicted. HL-60 cells were incubated without or with 20 nm PMA for 48 h, harvested, washed twice with PBS, and divided in samples of 2 × 105 cells/sample. The cells were then fixed (15 min at room temperature), washed three times with PBS, and then permeabilized (15 min at room temperature), using the Fixing and Permeabilization Kit (Caltag Laboratories, Burlingame, CA). During permeabilization, the cells were incubated with anti-human RPTPβ mouse monoclonal antibody (1:10) (Transduction Laboratories) or irrelevant mouse IgG antibodies (control) in PBS containing 2% BSA and 1 mm NaN3. Cells were then washed three times with the same buffer, followed by incubation with second antibody (1:400) (fluorescein isothiocyanate-labeled goat anti-mouse IgG; Amersham Pharmacia Biotech) for 30 min in the dark. After three more washings, the samples were analyzed using a FACScan flow cytometer (Becton Dickinson Immunocytometry System, La Jolla, CA) with excitation at 488 nm and emission at 529 nm. Phosphorothioate-capped oligonucleotides were synthesized by Amersham Pharmacia Biotech. The phosphorothioate-capped antisense oligonucleotide sequence was (positions 139–159) 5′-TsAsGsGsAsTsTsCsGsCsAsTsTsTsCsCsAsGsAsCsGs-3′; the corresponding sense oligonucleotide was 5′-CsGsTsCsTsGsGsAsAsAsTsGsCsGsAsAsTsCsCsTsAs-3′ (29.Krueger N.X. Saito H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7417-7421Crossref PubMed Scopus (201) Google Scholar). HL-60 cells were treated with the oligonucleotides (5, 10, or 15 μm) in 50 μl of RPMI medium supplemented with kanamycin, 50 μg/ml, in a 96-well plate (2 × 105 cells/well) and 6-cm Petri dishes for 3 h, followed by incubation with 20 nm PMA for 24 h (30.Eck S.L. Perkins N.D. Carr D.P. Nabel G.J. Mol. Cell. Biol. 1993; 13: 6530-6536Crossref PubMed Google Scholar). Cells used to assess vacuolating activity were then incubated with VacA for 8 h, and vacuolation was quantified in the neutral red assay. The cells used to assess RPTPβ expression were harvested and subjected to cell sorter analysis. For immunofluorescent staining, HL-60 cells were grown on 6-cm Petri dishes and stimulated with 20 nm PMA for 48 h. After incubation with 120 nm VacA for 1 h, the cells were washed twice with culture medium and centrifuged at 200 ×g for 5 min. The cells were smeared on glass slides and fixed in absolute methanol. After fixation, the cells were hydrated, incubated with a mixture of 10% normal goat serum/10% normal horse serum for 20 min at room temperature, and then incubated for 1 h at room temperature with a mixture of rabbit polyclonal antibody against VacA (1:500) and mouse monoclonal antibody against human RPTPβ (1:10). Slides were then incubated with the mixture of the two secondary antibodies (fluorescein isothiocyanate-conjugated horse anti-mouse IgG, diluted 1:100; Vector Laboratories and Texas Red-conjugated goat anti-rabbit IgG, diluted 1:100; Vector Laboratories) for 1 h at room temperature. Cells were examined with a confocal microscope (Bio-Rad MRC-600) equipped with argon and argon-krypton lasers for blue (488 nm) and green (544 nm) excitation. Negative control immunohistochemical procedures included: 1) omission of the primary antibody from the staining protocol, 2) omission of PMA stimulation of HL-60 cells, and 3) omission of VacA from the incubation with HL-60 cells. We used two PTPζ cDNA clones (pHPTPζ.fl.preUC and pSP65.HPTPζ256T) (29.Krueger N.X. Saito H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7417-7421Crossref PubMed Scopus (201) Google Scholar). A 1-μl sample of the 100× diluted cDNA pSP65.HPTPζ256T solution was used as template. Primers used for RPTPβ were: 5′-GGGCTAGCATGCGAATCCTAAAGCGTTTC-3′ (sense; positions 147–167) (GGGCTAGC was added to the primer to create a site for NheI) and 5′-ATATGGTCGGAAGAACAATGT-3′ (antisense; positions 1118–1138). AnXbaI site was located internally within the PCR products. PCR was performed in PCR Buffer II containing 1 mmMgCl2 and 1.25 units of Taq polymerase (Takara) in a final volume of 50 μl. Primers were added at a final concentration of 1 μm. Reactions were carried out in a DNA Thermal Cycler (Perkin-Elmer Cetus) for 35 cycles of 94 °C for 1 min, 50 °C for 2 min, and 72 °C for 2 min. PCR products of 994 bp were analyzed on ethidium bromide-stained 1% agarose gels. The PCR products were first subcloned into the pBluescript vector. The construct was then digested with NheI and XbaI to yield a 884-bp fragment. This subcloned fragment (884 bp) was then inserted into another vector, the pBK-CMV vector. Because the target sequence in the clones was too large, the pHPTPζ.fl.preUC clone was digested twice, first with XbaI and ClaI to yield a 4380-bp fragment and second with ClaI and SmaI to yield a 1988-bp fragment. The 4380-bp fragment was inserted first into the pBK-CMV vector with the 884-bp fragment followed by the last fragment (1988 bp) from pHPTPζ.fl.preUC for a total length of 7252 bp. BHK-21 cells in EMEM supplemented with 10% FCS and kanamycin (50 μg/ml) were cultured as monolayers for 24 h in an atmosphere of 5% CO2 at 37 °C. Cells were harvested with 10 mm Tris, 1 mm EDTA, pH 7.5 buffer, washed twice with serum-free EMEM, and suspended in EMEM; then 0.5 ml of this cell suspension was mixed with 30 μg of the RPTPβ-pBK-CMV plasmid. The cells and the plasmid were incubated for 10 min at room temperature before electroporation at 950 microfarad, 250 mV. The cuvettes were then placed in an ice bath for 10 min. The cells were then transferred to 10 ml of EMEM supplemented with 10% FCS and kanamycin (50 μg/ml), and 24 h later, cells were trypsinized and replated at a 1:10 dilution. After another 24 h, Geneticin (G418 sulfate; Life Technologies, Inc.) at a concentration of 800 μg/ml was added to the medium. After 5 days, transfected BHK-21 cell colonies were harvested for vacuolation assay and FACScan. After HL-60 cells were incubated with 5, 10, and 20 nm PMA for 24 h, their sensitivity to VacA was evaluated (Fig. 1). Addition of 120 nm VacA resulted in a remarkable vacuolation of HL-60 cells treated with 10 and 20 nm PMA, starting at 4 h post-exposure to toxin. No significant vacuolation was observed in cells treated with 0.1% (v/v) Me2SO or 5 nmPMA. HL-60 cells were exposed to 20 nm PMA for the indicated time before addition of 120 nm VacA, and vacuolation was assessed 8 h later. The longer the exposure of HL-60 cells to 20 nm PMA, the greater the vacuolation induced by VacA at 12 h than at 6 h (Fig. 2). More cells were adherent to the surface at 6 h. Cells treated with 0.1% (v/v) Me2SO exhibited negligible vacuolation in response to 120 nmVacA. Because RPTPβ was identified as a VacA receptor in AZ-521 cells,1 we investigated RPTPβ mRNA levels in HL-60 cells incubated without and with PMA by RNase Protection Assay (Fig.3). Treatment of HL-60 cells with 10 or 20 nm PMA for 24 and 48 h significantly increased RPTPβ mRNA. Measurable radioactivity was 49 and 67% for HL-60 cells treated with 10 and 20 nm PMA for 48 h, respectively, with AZ-521 cells as control (100%). In untreated and 0.1% Me2SO-treated HL-60 cells, RPTPβ mRNA was not detected, whereas AZ-521 cells expressed RPTPβ mRNA constitutively. These findings suggest that the exposure of HL-60 cells to 10 or 20 nm PMA for 24 and 48 h induced VacA sensitivity through enhancement of RPTPβ synthesis. To confirm the presence of RPTPβ protein, we performed a FACScan analysis using monoclonal anti-RPTPβ antibody (Fig.4). As shown in Fig. 4 B, PMA-stimulated HL-60 cells had greater (dotted lines) fluorescence than the control cells (solid line), indicating that the amount of RPTPβ was increased in cells treated with PMA. The positions of peak fluorescence in untreated HL-60 cells in assays using control and anti-RPTPβ antibodies were unchanged (Fig.4 A). To define more clearly the relationship between VacA sensitivity and expression of RPTPβ in PMA-treated HL-60 cells, RPTPβ sense or antisense oligonucleotides were added to the cells before exposure to PMA. As shown in Fig.5, there was a concentration-dependent inhibition of vacuolation in HL-60 cells treated for 3 h with the RPTPβ antisense oligonucleotide but not the sense oligonucleotide. The sense oligonucleotide-treated cells had the same degree of vacuolation as the controls (0.1% (v/v) Me2SO-treated cells without oligonucleotide). This result suggests that RPTPβ specifically functions as the primary functional receptor for VacA in HL-60 cells treated with PMA. To characterize further RPTPβ expression in HL-60 cells incubated with RPTPβ antisense oligonucleotides, cell sorter analysis was performed to show the dose-dependent inhibition of expression of RPTPβ. In Fig.6 A, when PMA-stimulated HL-60 cells were treated with RPTPβ sense oligonucleotides, no visible shift in the amount of fluorescence was seen, whereas cells that were treated with antisense oligonucleotide exhibited decreased fluorescence inversely proportional to the concentration of RPTPβ antisense oligonucleotide. To demonstrate the colocalization of VacA and RPTPβ on the surface of HL-60 cells treated with PMA, we carried out double-immunostaining by confocal microscopy using anti-VacA and anti-RPTPβ antibodies (Fig.7). In HL-60 cells treated with 20 nm PMA for 48 h followed by incubation with 120 nm VacA for 1 h, RPTPβ and VacA were extensively colocalized at the cytoplasmic membrane. In many cells, the red fluorescence of VacA rimmed the yellow, which resulted from superimposition of green indicating RPTPβ (Fig. 7 A). RPTPβ was found in the cytoplasm in addition to its primary location at the plasma membrane (Fig. 7 B). The VacA was similarly located in the cytoplasm as well as in the plasma membrane (Fig.7 C). In untreated HL-60 cells (i.e. without exposure to PMA or VacA), no fluorescence indicative of RPTPβ or VacA was observed (Fig. 7 D). HL-60 cells were incubated for 24 h at 37 °C with 20 nm PMA to induce differentiation to macrophage-like cells, with 1.2% (v/v) Me2SO or 100 nm retinoic acid to induce granulocyte-like differentiation, or with 10 nmVitD3 or IFN-γ (100 units/ml) to induce monocyte-like cells. The cells were then incubated with 120 nm VacA for an additional 8 h at 37 °C (Fig.8). VacA-induced vacuolation similar to that seen after PMA was clearly observed in HL-60 cells treated with VitD3 or IFN-γ. No significant vacuolation was detected in HL-60 cells treated with 1.2% (v/v) Me2SO or retinoic acid. Limited but significant expression of RPTPβ mRNA was observed in HL-60 cells treated with VitD3 (28%), IFN-γ (48%), and PMA (61%) with RNase Protection Assay (Fig.9) as compared with AZ-521 cells. Me2SO and retinoic acid, however, which stimulate the maturation of HL-60 into granulocyte-like cells, did not induce VacA sensitivity or increase expression of RPTPβ mRNA.Figure 9Effect of differentiating agents on RPTPβ mRNA expression in HL-60 cells.mRNA was harvested after cells were incubated with the indicated agents for 24 h. RNase-protected RNA fragments were analyzed using 5% polyacrylamide/8 m urea gels and autoradiography.Lane 1, RNA probes/(−) RNase; lane 2, RNA probes/(+) RNase; lane 3, AZ-521 cells (positive control). The other lanes are HL-60 cells without additions (negative control;lane 4), with 1.2% (v/v) Me2SO (lane 5), with 100 nm retinoic acid (lane 6), with 10 nm VitD3 (lane 7), with IFN-γ (100 units/ml; lane 8), and with20 nm PMA (24 h;lane 9). Data are representative of three separate experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) BHK-21 cells that were transfected with RPTPβ exhibited greater sensitivity to VacA than did untransfected cells or cells transfected with the vector only (Fig.10 A). Confirmation of the newly expressed RPTPβ itself in the cell membrane was seen upon cell sorting analysis. There was a significant shift in the fluorescence of the cells (clone 26) transfected with RPTPβ as compared with the control (untransfected cells) and cells transfected with the vector only (Fig. 10 B). VacA has been implicated as one of the major pathogenic factors inH. pylori-induced disease. VacA has been found mainly in the gastric mucosa, and its ability to cause vacuolation has been widely documented in many cell types, with the notable exception of HL-60 (6.Cover T.L. Trends Microbiol. 1998; 6: 127-128Abstract Full Text Full Text PDF PubMed Google Scholar). de Bernard et al. (19.de Bernard M. Moschioni M. Papini E. Telford J.L. Rappuoli R. Montecucco C. FEBS Lett. 1998; 436: 218-222Crossref PubMed Scopus (14) Google Scholar) recently reported that PMA induced VacA susceptibility in HL-60 cells and increased the sensitivity of HeLa cells to VacA. The increased sensitivity of HeLa cells to VacA required protein kinase C activation for 10–15 h, suggesting that it resulted from new protein synthesis. Because we purified a putative receptor for VacA from AZ-521 cells and identified it as RPTPβ,1 we investigated the association between differentiation of HL-60 cells induced with several agents and RPTPβ expression or VacA sensitivity to determine whether RPTPβ indeed serves as the primary functional receptor for VacA. We also attempted to express RPTPβ in BHK-21 cells, another cell line that is resistant to VacA. We now report that VacA sensitivity clearly parallels induction of RPTPβ mRNA expression by PMA (Fig. 3). In HL-60 cells both induction of VacA sensitivity and expression of RPTPβ mRNA were directly dependent on the concentration and time of exposure of cells to PMA. In addition to enhanced RPTPβ mRNA expression in HL-60 cells treated with PMA, an increased amount of RPTPβ protein was demonstrated using indirect immunofluorescence. The mean fluorescence per cell of RPTPβ was clearly greater after PMA treatment, which did not alter the fluorescence of cells reacted with nonspecific antibodies, indicating the presence of newly expressed surface RPTPβ (Fig. 4). Thus, PMA substantially increased both RPTPβ mRNA and protein in HL-60 cells, strongly suggesting that the acquisition of VacA sensitivity of HL-60 cells induced by PMA resulted from newly expressed RPTPβ. This hypothesis was further examined by assessing the effects of RPTPβ antisense oligonucleotide on the induction of the VacA sensitivity by PMA and on the expression of RPTPβ protein (Figs. 5and 6) as well as by colocalization of VacA and RPTPβ in PMA-treated HL-60 cells (Fig. 7). Treatment of HL-60 cells with RPTPβ antisense oligonucleotide before incubation with PMA led to the dose-dependent decrease in VacA sensitivity, whereas RPTPβ sense oligonucleotide was without effect (Fig. 5). Antisense oligonucleotide also decreased RPTPβ expression (by FACScan) in a dose-dependent manner, whereas sense oligonucleotide had no effect (Fig. 6). Confocal microscopy revealed that RPTPβ was mainly located near the plasma membrane with some in the cytoplasm. Both RPTPβ and VacA colocalized to a significant extent in the two regions. VacA was also found outside of the region of colocalization with RPTPβ, possibly on the exterior of the cell. The latter may represent toxin bound but not yet internalized. The site of localization of VacA with RPTPβ in the cytoplasm is unclear; it may represent an endosomal structure involved in toxin processing. Numerous agents have been used to induce differentiation of HL-60 cells (18.Collins S. Blood. 1987; 70: 1233-1244Crossref PubMed Google Scholar). PMA inhibits cell proliferation and promotes cell adhesion, spreading, and phagocytic activity, all characteristic of macrophage-like cells (14.Huberman E. Callaham M. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 1293-1297Crossref PubMed Scopus (490) Google Scholar, 15.Rovera G. Santoli D. Damsky C. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 2779-2783Crossref PubMed Scopus (870) Google Scholar). In this study, consistent with published findings (14.Huberman E. Callaham M. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 1293-1297Crossref PubMed Scopus (490) Google Scholar), almost 80% of cultured HL-60 cells became adherent to the surface after 24 h of incubation with 10 nm PMA. Retinoic acid and Me2SO at a concentration that causes development into granulocytic cells failed to induce RPTPβ expression (Fig. 9) or VacA sensitivity (Fig. 8). Because PMA is usually grouped with VitD3 and IFN-γ among reagents that stimulate differentiation of HL-60 cells into monocytic/macrophage-like cells, the latter were tested for their ability to induce VacA sensitivity. Like PMA, both agents induced VacA sensitivity and increased expression of RPTPβ mRNA and protein in HL-60 cells. RPTPβ cDNA was also stably expressed in BHK-21 cells. Cells expressing RPTPβ (clone 26) showed greater sensitivity to VacA on the neutral red uptake assay than did control cells or those transfected with vector only (Fig. 10 A). Presence of the newly expressed RPTPβ was confirmed on FACScan analysis by the shifting of the curve to the right for BHK-21 clone 26 (Fig. 10 B). Thus, we have proven that expression of RPTPβ cDNA confers VacA sensitivity on both HL-60 cells during PMA-induced differentiation to macrophage/monocytic-like cells and BHK-21 cells transfected with the RPTPβ cDNA. This further supports our hypothesis that RPTPβ acts as the receptor for VacA. RPTPβ is most abundant in cells of nervous system where it regulates the maturation, development, and differentiation of neuronal and glial cells (31.Holland S. Peles E. Pawson T. Schelssinger J. Curr. Opin. Neurobiol. 1998; 8: 117-127Crossref PubMed Scopus (106) Google Scholar). It is postulated that phosphorylation (32.Meighan-Mantha R.L. Wellstein A. Riegel A.T. Exp. Cell Res. 1997; 234: 321-328Crossref PubMed Scopus (17) Google Scholar, 33.Juan G. Li X. Darzynkiewicz Z. Exp. Cell Res. 1998; 244: 83-92Crossref PubMed Scopus (33) Google Scholar, 34.Simpson R.U. O'Connell T.D. Pan Q. Newhouse J. Somerman M.J. J. Biol. Chem. 1998; 273: 19587-19591Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 35.Korchak H.M. Rossi M.W. Kilpatrick L.E. J. Biol. Chem. 1998; 273: 27292-27299Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar) and dephosphorylation (20.Tanuma N. Nakamura K. Kikuchi K. Eur. J. Biochem. 1999; 259: 46-54Crossref PubMed Scopus (51) Google Scholar, 36.Walton K. Dixon J. Annu. Rev. Biochem. 1993; 62: 101-120Crossref PubMed Scopus (415) Google Scholar, 37.Kasugai I Morimoto K. Hayakawa T. Cancer Lett. 1997; 120: 223-227Crossref PubMed Scopus (2) Google Scholar, 38.Elson A. Leder P. Proc. Natl. Acad., Sci. U. S. A. 1995; 92: 12235-12239Crossref PubMed Scopus (75) Google Scholar) of proteins are important in signaling the maturation of HL-60 cells by differentiation-promoting reagents. Protein tyrosine phosphatase, PTP1C, has been reported to play an important role in suppressing the proliferation of hematopoietic cells and inducing cellular maturation (36.Walton K. Dixon J. Annu. Rev. Biochem. 1993; 62: 101-120Crossref PubMed Scopus (415) Google Scholar). The high expression of PTP1C in HL-60 cells appears to be involved in the enhancement of susceptibility to macrophage-like differentiation by PMA (37.Kasugai I Morimoto K. Hayakawa T. Cancer Lett. 1997; 120: 223-227Crossref PubMed Scopus (2) Google Scholar). On the other hand, in the early stages of the differentiation of HL-60 cells by PMA, the promoter activity of the 5′-flanking region of cytosolic, but not that of transmembrane RPTPε, was dramatically elevated, and cytosolic RPTPε mRNA was highly expressed, suggesting its involvement in the events leading to differentiation of HL-60 cells (20.Tanuma N. Nakamura K. Kikuchi K. Eur. J. Biochem. 1999; 259: 46-54Crossref PubMed Scopus (51) Google Scholar, 38.Elson A. Leder P. Proc. Natl. Acad., Sci. U. S. A. 1995; 92: 12235-12239Crossref PubMed Scopus (75) Google Scholar). RPTPβ may also promote maturation of HL-60 cells into a monocytic/macrophage phenotype. The exact mechanism by which vacuolization is related to signaling from RPTPβ remains to be determined. Interaction of VacA with RPTPβ may serve to bind the toxin to the cell surface and promote internalization. Alternatively, VacA may affect the activity of RPTPβ and then facilitate its entrance leading to vacuolation of the cells. We thank I. Kato (Medical School of Chiba University), M. Nakamura (Institute of Tropical Medicine, Nagasaki University), Z.-X. Yu and V. J. Ferrans (NHLBI, National Institutes of Health) for helpful discussions, and M. Vaughan (NHLBI, National Institutes of Health) for critical review of the manuscript." @default.
• W2126451227 created "2016-06-24" @default.
• W2126451227 creator A5012263872 @default.
• W2126451227 creator A5013280974 @default.
• W2126451227 creator A5024673298 @default.
• W2126451227 creator A5030442592 @default.
• W2126451227 creator A5033869888 @default.
• W2126451227 creator A5047904444 @default.
• W2126451227 creator A5048042168 @default.
• W2126451227 creator A5057900466 @default.
• W2126451227 creator A5059148796 @default.
• W2126451227 creator A5063485351 @default.
• W2126451227 creator A5065566018 @default.
• W2126451227 creator A5079191505 @default.
• W2126451227 creator A5088648351 @default.
• W2126451227 date "2000-05-01" @default.
• W2126451227 modified "2023-10-01" @default.
• W2126451227 title "Morphologic Differentiation of HL-60 Cells Is Associated with Appearance of RPTPβ and Induction of Helicobacter pyloriVacA Sensitivity" @default.
• W2126451227 cites W1500891430 @default.
• W2126451227 cites W1522153085 @default.
• W2126451227 cites W1621166403 @default.
• W2126451227 cites W1773256867 @default.
• W2126451227 cites W1980612524 @default.
• W2126451227 cites W1986878477 @default.
• W2126451227 cites W1996249316 @default.
• W2126451227 cites W1999689481 @default.
• W2126451227 cites W2002549957 @default.
• W2126451227 cites W2014129580 @default.
• W2126451227 cites W2014520088 @default.
• W2126451227 cites W2018138643 @default.
• W2126451227 cites W2034190134 @default.
• W2126451227 cites W2037871535 @default.
• W2126451227 cites W2042182894 @default.
• W2126451227 cites W2044854718 @default.
• W2126451227 cites W2045961534 @default.
• W2126451227 cites W2046748571 @default.
• W2126451227 cites W2047718649 @default.
• W2126451227 cites W2052561467 @default.
• W2126451227 cites W2058901530 @default.
• W2126451227 cites W2068918042 @default.
• W2126451227 cites W2070456887 @default.
• W2126451227 cites W2080387736 @default.
• W2126451227 cites W2081910096 @default.
• W2126451227 cites W2088308620 @default.
• W2126451227 cites W2096372770 @default.
• W2126451227 cites W2118522916 @default.
• W2126451227 cites W2122734844 @default.
• W2126451227 cites W2132293085 @default.
• W2126451227 cites W2132867417 @default.
• W2126451227 cites W2141083396 @default.
• W2126451227 cites W2144907122 @default.
• W2126451227 cites W2155237708 @default.
• W2126451227 cites W2160421139 @default.
• W2126451227 cites W4211017205 @default.
• W2126451227 cites W4293247451 @default.
• W2126451227 doi "https://doi.org/10.1074/jbc.275.20.15200" @default.
• W2126451227 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/10809755" @default.
• W2126451227 hasPublicationYear "2000" @default.
• W2126451227 type Work @default.
• W2126451227 sameAs 2126451227 @default.
• W2126451227 citedByCount "75" @default.
• W2126451227 countsByYear W21264512272012 @default.
• W2126451227 countsByYear W21264512272013 @default.
• W2126451227 countsByYear W21264512272014 @default.
• W2126451227 countsByYear W21264512272015 @default.
• W2126451227 countsByYear W21264512272016 @default.
• W2126451227 countsByYear W21264512272017 @default.
• W2126451227 countsByYear W21264512272018 @default.
• W2126451227 countsByYear W21264512272019 @default.
• W2126451227 countsByYear W21264512272020 @default.
• W2126451227 countsByYear W21264512272022 @default.
• W2126451227 countsByYear W21264512272023 @default.
• W2126451227 crossrefType "journal-article" @default.
• W2126451227 hasAuthorship W2126451227A5012263872 @default.
• W2126451227 hasAuthorship W2126451227A5013280974 @default.
• W2126451227 hasAuthorship W2126451227A5024673298 @default.
• W2126451227 hasAuthorship W2126451227A5030442592 @default.
• W2126451227 hasAuthorship W2126451227A5033869888 @default.
• W2126451227 hasAuthorship W2126451227A5047904444 @default.
• W2126451227 hasAuthorship W2126451227A5048042168 @default.
• W2126451227 hasAuthorship W2126451227A5057900466 @default.
• W2126451227 hasAuthorship W2126451227A5059148796 @default.
• W2126451227 hasAuthorship W2126451227A5063485351 @default.
• W2126451227 hasAuthorship W2126451227A5065566018 @default.
• W2126451227 hasAuthorship W2126451227A5079191505 @default.
• W2126451227 hasAuthorship W2126451227A5088648351 @default.
• W2126451227 hasBestOaLocation W21264512271 @default.
• W2126451227 hasConcept C127413603 @default.
• W2126451227 hasConcept C153911025 @default.
• W2126451227 hasConcept C185592680 @default.
• W2126451227 hasConcept C21200559 @default.
• W2126451227 hasConcept C24326235 @default.
• W2126451227 hasConcept C2776409635 @default.
• W2126451227 hasConcept C2780832600 @default.
• W2126451227 hasConcept C54355233 @default.
• W2126451227 hasConcept C86803240 @default.
• W2126451227 hasConcept C89423630 @default.
• W2126451227 hasConcept C95444343 @default.

• next