[N-acetyl-L-cysteine inhibits adenoviral E1A-involved transactivation of nuclear factor-kappaB in rat alveolar epithelial cells.].
OBJECTIVE
the relationship between latent adenovirus infection and airway inflammation had not been well documented. The aim of this study was to illustrate the roles of adenovirus E1A protein on the transactivation of NF-kappaB, AP-1 in response to inflammatory stimuli and the effect of N-Acetylcysteine (NAC) upon the transactivation of NF-kappaB and AP-1 in cells stably expressing E1A protein.


METHODS
rat alveolar epithelial cells stably expressing adenoviral E1A or control plasmid were developed. For isolation of nuclear extracts, 5 x 10(5) cells were plated and grown overnight in 60 mm dishes. Experiments were repeated 3 times. The cell model of stably expressing adenoviral E1A was stimulated by LPS or TNF-alpha and treated with NAC, a precursor for cysteine. The NF-kappaB and AP-1 transcriptional activity were measured by LUC report system. The expression of NF-kappaB and AP-1 were measured by Western blot. Differences between groups were assessed for significance by Student' t test, and multiple comparisons were made by one-way ANOVA.


RESULTS
the luciferase activity derived by NF-kappaB element was (9 698 +/- 98) RLU in untreated E1A-positive clones and (101 195 +/- 234), and (170 385 +/- 443) RLU in LPS and TNF-alpha-stimulated cells, which were significantly higher than that of the control group 2 077 +/- 107, 67 846 +/- 332, 95 743 +/- 211 respectively. The luciferase activity derived by AP-1 element was 9 034 +/- 78 RLU in untreated E1A-positive clones and 26 343 +/- 398 and 31 731 +/- 332 RLU in LPS and TNF-alpha-stimulated cells, which were significantly higher than that of the control group 2 845 +/- 93, 10 772 +/- 432, 11 005 +/- 556 respectively. The densitometry of the NF-kappaB expression in E1A-positive clones were 79.3 +/- 4.6 and 80.3 +/- 3.8 respectively without treatment and were 81.8 +/- 3.9 - 89.9 +/- 1.6 and 94.1 +/- 1.9 to 99.8 +/- 1.6 respectively under LPS or TNF-alpha stimulation, which were significantly higher than that of the control group (68.3 +/- 3.8, 69.4 +/- 4.3 respectively) without stimulation and 70.1 +/- 2.8 to 80.8 +/- 3.6, 73.4 +/- 4.9 to 83.2 +/- 6.7 respectively under stimulation. The level of AP-1 expression did not show difference upon treatment with LPS or TNF-alpha in either cell clones. The densitometry of the NF-kappaB expression in E1A-positive clones were 3.2 +/- 0.1 and 3.3 +/- 0.1 respectively under LPS and TNF-alpha-stimulation and 1.98 +/- 0.2 and 1.9 +/- 0.2 respectively upon treatment for LPS and TNF-alpha with NAC pre-incubation.


CONCLUSIONS
these results indicate that E1A protein upregulated NF-kappaB transcription activity induced by LPS and TNF-alpha in rat alveolar epithelial cells and this effect could be repressed by NAC. The mechanisms underlying transactivation of NF-kappaB involved by E1A may be related to oxidative stress.
Chen
Juan
J
The State Key Lab of Respiratory Disease, Guangzhou Institute of Respiratory Disease, the First Affiliated Hospital, Guangzhou Medical University, Guangzhou 510120, China.
Li
Bing
B
Ran
Pi-Xin
PX
D000111:Acetylcysteine / Q000494:pharmacology*; D000256:Adenoviridae / Q000235:genetics; D017500:Adenovirus E1A Proteins / Q000494:pharmacology*; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology*; D002460:Cell Line; D004847:Epithelial Cells; D016328:NF-kappa B / Q000378:metabolism*; D011650:Pulmonary Alveoli / Q000166:cytology; D051381:Rats; D015533:Transcriptional Activation
D017500:Adenovirus E1A Proteins; D000975:Antioxidants; D016328:NF-kappa B; D000111:Acetylcysteine