Helicobacter pylori inhibits expression of heat shock protein 70 (HSP70) in human epithelial cell line. Importance of Cag A protein.
Heat shock proteins (HSP) are crucial for the maintenance of cell integrity under normal cell growth and at pathophysiological conditions such as colonization of gastric mucosa by Helicobacter pylori (Hp). The effect of Hp on mRNA expression for HSP70 in the gastric epithelial cells in vitro has been little studied and remains inconclusive. In this study we attempted to determine the alterations in gene expression for HSP70 induced by two live strains of Hp in the epithelial MKN7 cells. The following Hp strains were employed; 1) Hp strain expressing cagA and vacA, and 2) cagA and vacA negative Hp strain without or with addiction of exogenous recombinant protein CagA. MKN7 cells were incubated in a standard medium RPMI 1640 supplemented with 10% fetal bovine serum at 37 degrees C with 5% CO2 and humidified atmosphere under basal condition or in a presence of Hp (1 x 10(9) CFU per dish) without or with the recombinant CagA (10 microg/ml of RPMI 1640 medium). After 3 h, 24 h and 48 h of incubation with Hp and in some experiments with the prolonged incubation time up to 72 h, the cells were harvested, the total cellular RNA was isolated and the expression of mRNA for HSP70 was determined by RT-PCR. The incubation of the MKN cells with CagA protein alone failed to affect significantly the expression of HSP70. In contrast, the strain Hp (cagA+, vacA+) inhibited in time-dependent manner the expression of mRNA for HSP70. When the MKN7 cells were coincubated with Hp (cagA+, vacA+) and exogenous CagA, the significant inhibition of the signal intensity for HSP70 mRNA was observed at 3 h and 24 h of incubation and these effects were followed by complete disappearance of the signal for HSP70 mRNA at 48 h. The incubation of MKN7 with Hp (cagA-, vacA-) also significantly attenuated the expression of HSP70 mRNA with the most pronounced inhibitory effect observed at 72 h of incubation with this Hp strain. Addition of the recombinant CagA to Hp (cagA-, vacA-) completely suppressed the expression of HSP70 at 48 h and 72 h after the end of incubation periods. We conclude that 1) both, Hp (cagA+, vacA+) and Hp (cagA-, vacA-) inhibit expression of HSP70 in MKN7 human gastric epithelial cells independently of the presence or absence of cagA gene, and that 2) recombinant CagA protein may exert biological activity in vitro via acceleration of inhibitory effect of Hp negative for Cag A and VacA on HSP70 expression in epithelial cells infected with this bacteria.
Targosz
A
A
Department of Physiology Jagiellonian University Medical College, Cracow, Poland.
Pierzchalski
P
P
Krawiec
A
A
Szczyrk
U
U
Brzozowski
T
T
Konturek
S J
SJ
Pawlik
W W
WW
D000942:Antigens, Bacterial / Q000235:genetics / Q000494:pharmacology / Q000502:physiology; D001426:Bacterial Proteins / Q000235:genetics / Q000494:pharmacology / Q000502:physiology; D045744:Cell Line, Tumor; D004847:Epithelial Cells / Q000378:metabolism / Q000382:microbiology / Q000473:pathology; D015870:Gene Expression / Q000187:drug effects / Q000235:genetics*; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics*; D016480:Helicobacter pylori / Q000235:genetics / Q000254:growth & development*; D006801:Humans; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013274:Stomach Neoplasms / Q000235:genetics / Q000382:microbiology / Q000473:pathology; D013997:Time Factors
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D018840:HSP70 Heat-Shock Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; C074689:VacA protein, Helicobacter pylori; C080866:cagA protein, Helicobacter pylori