Journal
ANALYTICAL BIOCHEMISTRY
Volume 430, Issue 1, Pages 92-96Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2012.08.003
Keywords
Bioluminescent; Cell-based assay; Cholesterol 7 alpha-hydroxylase; Reporter gene; Chlamydia pneumoniae; Human cytomegalovirus
Funding
- Ministry for Education, Universities, and Research (MIUR, Italy) [PRIN 2009MB4AYL, FIRB 2008 RBFR08SZTR]
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Chlamydia pneumoniae and human cytomegalovirus (HCMV) are intracellular pathogens able to infect hepatocytes, causing an increase in serum triglycerides and cholesterol levels due to the production of inflammatory cytokines. We investigated whether these pathogens could interfere with cholesterol metabolism by affecting activity of hepatic cholesterol 7 alpha-hydroxylase (CYP7A1) promoter. CYP7A1 is the rate-limiting enzyme responsible for conversion of cholesterol to bile acids, which represents the main route of cholesterol catabolism. A straightforward dual-reporter bioluminescent assay was developed to simultaneously monitor CYP7A1 transcriptional regulation and cell viability in infected human hepatoblastoma HepG2 cells. C pneumoniae and HCMV infection significantly decreased CYP7A1 promoter activity in a dose-dependent manner, with maximal inhibitions of 33 +/- 10% and 32 +/- 4%, respectively, at a multiplicity of infection of 1. To support in vitro experiments, serum cholesterol, high-density lipoprotein (HDL) cholesterol, triglycerides and glucose levels were also measured in Balb/c mice infected with C. pneumoniae. Serum cholesterol and triglycerides also increased in infected mice compared with controls. Although further investigation is required, this work presents the first experimental evidence that C. pneumoniae and HCMV inhibit CYP7A1 gene transcription in the cultured human hepatoblastoma cell line. (c) 2012 Elsevier Inc. All rights reserved.
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