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Zhonghua Jie He He Hu Xi Za Zhi. 2010 Jul;33(7):500-4.

[The relationship between cyclic adenosine monophosphate response element-binding protein and interferon-γ gene promoter in tuberculosis patients].

[Article in Chinese]

Author information

  • 1Department of Molecular Biology, Beijing Tuberculosis and Thoracic Tumor Research Institute, Beijing 101149, China.

Abstract

OBJECTIVE:

To study the relationship between cAMP response element binding protein (CREB) and the interferon-γ (IFN-γ) proximal promoter in patients with tuberculosis.

METHODS:

CD₃(+) T cells were isolated from 25 pulmonary tuberculosis patients, who had been treated in Beijing Chest Hospital from January to December 2007, and 18 PPD-positive healthy donors. After extraction of nuclear proteins, electrophoretic mobility shift assay (EMSA) was performed to determine nuclear protein binding to the IFN-γ proximal promoter in vitro, and the specificity of binding complex was tested by competitive EMSA. Chromatin immunoprecipitation (ChIP) with anti-CREB Ab was used to determine whether CREB binded to the IFN-γ proximal promoter in vivo in live T cells exposed to microbial Ags. Western blotting with anti-CREB Ab was performed to compare the expression level of CREB in tuberculosis patients and PPD-positive healthy donors. Western blotting with Abs specific for serine 133-phosphorylated CREB was performed to determine whether M.tuberculosis Ags elicited phosphorylation of CREB.

RESULTS:

The results of EMSA showed a low-mobility complex binding to the IFN-γ promoter, and the binding pattern observed was similar for T cells from all 18 PPD-positive healthy donors. However, for T cells from 18 of 25 tuberculosis patients, the low-mobility complex was absent. The results of competitive EMSA showed that these nuclear proteins specifically bound to the IFN-γ promoter region and contained CREB. The results of ChIP showed a 204 bp band yielded in CD₃(+) T cells from 10 PPD-positive healthy donors, but 12 tuberculosis patients didn't yield the band. CREB expression markedly decreased in tuberculosis patients compared with healthy donors detected by Western blotting. Furthermore, M. tuberculosis Ags also elicited phosphorylation of CREB in CD₃(+) T cells from PPD-positive healthy donors, but not in CD₃(+) T cells from tuberculosis patients.

CONCLUSIONS:

CREB protein binding to IFN-γ proximal promoter was reduced in tuberculosis patients compared with healthy donors. Tuberculosis patients had diminished CREB protein levels, and reduced ability of binding to the IFN-γ promoter.

PMID:
20979795
[PubMed - indexed for MEDLINE]
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