Archives

  • 2018-07
  • 2019-07
  • 2019-08
  • 2019-09
  • 2019-10
  • 2019-11
  • 2020-03
  • 2020-07
  • 2020-08
  • 2021-03
  • cells infected with H pylori G DcagPAI Figure

    2020-08-28

    489 cells infected with H pylori G27 DcagPAI (Figure 2E). overexpressing AGS SR 11302 infected with H pylori and, conse- 548
    quently, H pylori–mediated induction of CD44 expression was 549
    491 Increased Expression of ESRP1 and b-Catenin in suppressed significantly (Figure 3D). By contrast, 550
    492 CCT031374 treatment did not attenuate the increase 551
    CAPZA1-Overexpressing Cells, in Combination
    494 With Accumulation of CagA, Induces CD44v9 subsequently examined ESRP1 expression in CAPZA1- 553
    495 Expression overexpressing AGS cells treated with CCT031374. Treat- 554
    496 Translocated CagA was strongly detected in CAPZA1- ment with CCT031374 scarcely affected the high expression 555
    497 overexpressing cells infected with H pylori G27 and was of ESRP1 in CAPZA1-overexpressing AGS cells (Figure 3E). 556
    498 not detected in H pylori G27-infected AGS cells or H pylori Based on these results, we hypothesized that up-regulation of 557
    499 G27 DcagPAI-infected cells, confirming that translocated CD44v9 expression in CAPZA1-overexpressing cells infected 558
    500 CagA accumulates in CAPZA1-overexpressing AGS cells with H pylori is mediated mainly by ESRP1. To test this hy- 559
    501 (Figure 3A). Overexpression of CAPZA1 did not induce pothesis, we assessed the effect of ESRP1 knockdown on up- 560
    503 pylori G27 infection did not induce CD44v9 expression in AGS effects of the small interfering RNA (siRNA), we used 2 562
    505 infection significantly enhanced CD44v9 expression in siRNA 2). ESRP1 knockdown attenuated the up-regulation of 564
    506 CAPZA1-overexpressing AGS cells (Figure 3A, lanes 2 and 4). CD44v9 expression in CAPZA1-overexpressing AGS cells 565
    507 Expression of CD44s in CAPZA1-overexpressing AGS cells infected with H pylori in comparison with control siRNA 566
    508 was higher than that in AGS cells and was increased further (Figure 3F). These results indicate that up-regulation of 567
    509 by H pylori G27 infection (Figure 3A). CD44v9 is generated by CD44v9 expression in CAPZA1-overexpressing cells is owing 568
    510 alternative splicing of CD44 mediated by ESRP.23 Intrigu- mainly to increased expression of ESRP1, but not of b-cat- 569
    511 ingly, basal ESRP1 expression was higher in CAPZA1- enin. Collectively, our findings suggest that CAPZA1 over- 570
    512 overexpressing AGS cells than in AGS cells (Figure 3A, lanes expression predisposes cells to develop into CD44v9-positive 571
    513 1 and 2). CD44 is a target gene of nuclear b-catenin.24 Basal cells via accumulation of CagA upon H pylori infection. 572
    516 Figure 2. (See previous page). Relationship between CD44v9 and CAPZA1 expression upon H pylori infection. (A) 575
    517 MKN28 cells were transfected with the pRC/CMV-CD44s or pRC/CMV-CD44v expression plasmid, and then CAPZA1 mRNA 576
    518 expression was determined by real-time quantitative PCR. Data are presented as the means ± SD of 3 independent assays. P 577
    values were calculated by a 1-way analysis of variance. (B) Expression of CAPZA1 in CD44s- and CD44v9-expressing MKN28
    cells. (C) AGS cells were transfected with pCMV-ctrl (CAPZA1 overexpression [-]) or pCMV-CAPZA1 (CAPZA1 overexpression
    [þ]), infected with H pylori G27 or H pylori G27 DcagPAI for 5 hours (multiplicity of infection, 50), and incubated in antibiotic-
    containing medium for 24 hours. mRNA expression of CD44v9 was measured by real-time qPCR. Data are presented as the
    523 of variance (right panel). (D) MKN28 cells were transfected with pTet-off-ctrl (MKN-II cells) or pTet-off-cagA (WT-A10 cells). 582 524 CagA expression in WT-A10 cells was induced by culture in the absence of doxycycline for 24 hours. mRNA expression of 583
    CD44v9 and CAPZA1 was determined by real-time qPCR. Data are presented as the means ± SD of 3 independent assays. *P
    < .01. P values were calculated by a 1-way analysis of variance. (E) AGS cells were transfected with pCMV-ctrl or pCMV-
    CAPZA1, infected with H pylori G27 or H pylori G27 DcagPAI for 5 hours (multiplicity of infection, 50), and incubated in
    antibiotic-containing medium for 24 hours. mRNA expression of SALL4 and KLF5 was determined by real-time qPCR. Data are
    6 Tsugawa et al
    Cellular and Molecular Gastroenterology and Hepatology Vol. -, No. -
    CAPZA1 Enhance CD44v9 Expression 7
    707 H pylori Infection–Induced Oxidative Stress
    polymerase chain reaction (qPCR) primers that specifically 766
    708 Increases CAPZA1 Expression in the Gastric
    targeted 1 kb downstream of the transcription start site of 767
    CAPZA1. The level of acetylated histone H3 was increased