ABSTRACT
The etiology of human female infertility is often uncertain. The sterility of high-density lipoprotein (HDL) receptor-negative (SR-BI(-/-)) female mice suggests a link between female infertility and abnormal lipoprotein metabolism. SR-BI(-/-) mice exhibit elevated plasma total cholesterol [with normal-sized and abnormally large HDL and high unesterified to total plasma cholesterol (UC:TC) ratio]. We explored the influence of hepatic SR-BI on female fertility by inducing hepatic SR-BI expression in SR-BI(-/-) animals by adenovirus transduction or stable transgenesis. For transgenes, we used both wild-type SR-BI and a double-point mutant, Q402R/Q418R (SR-BI-RR), which is unable to bind to and mediate lipid transfer from wild-type HDL normally, but retains virtually normal lipid transport activities with low-density lipoprotein. Essentially wild-type levels of hepatic SR-BI expression in SR-BI(-/-) mice restored to nearly normal the HDL size distribution and plasma UC:TC ratio, whereas approximately 7- to 40-fold overexpression dramatically lowered plasma TC and increased biliary cholesterol secretion. In contrast, SR-BI-RR overexpression had little effect on SR-BI(+/+) mice, but in SR-BI(-/-) mice, it substantially reduced levels of abnormally large HDL and normalized the UC:TC ratio. In all cases, hepatic transgenic expression restored female fertility. Overexpression in SR-BI(-/-) mice of lecithin:cholesterol acyl transferase, which esterifies plasma HDL cholesterol, did not normalize the UC:TC ratio, probably because the abnormal HDL was a poor substrate, and did not restore fertility. Thus, hepatic SR-BI-mediated lipoprotein metabolism influences murine female fertility, raising the possibility that dyslipidemia might contribute to human female infertility and that targeting lipoprotein metabolism might complement current assisted reproductive technologies.
Subject(s)
Fertility , Lipoproteins, HDL/metabolism , Liver/metabolism , Scavenger Receptors, Class B/physiology , Adenoviridae/genetics , Animals , Cholesterol/metabolism , Female , Mice , Mice, Inbred C57BL , Phosphatidylcholine-Sterol O-Acyltransferase/metabolism , Scavenger Receptors, Class B/geneticsABSTRACT
BACKGROUND: Recombinant adenoviruses are an established tool for somatic gene transfer to multiple cell types in animals as well as in tissue culture. However, generation of adenoviruses expressing transgenes that are potentially toxic to the host cell line represents a practical problem. The aim of this study was to construct an adenoviral expression system that prevents transgene expression during the generation and propagation of the virus, and allows efficient gene transfer to lung and liver, major target organs of gene therapy. METHODS: Using the tet-off system we constructed tetracycline (tet) regulatable recombinant adenoviruses expressing the marker gene LacZ (Adtet-off.LacZ) as well as a secretory protein, human group IIA secretory phospholipase A(2) (Adtet-off.hsPLA(2)). Expression (Western blot, activity assay) was tested in vitro (HeLa cells), and in vivo by gene transfer to lung and liver. RESULTS: Without addition of tetracycline we demonstrated expression of LacZ (Adtet-off.LacZ) and sPLA(2) (Adtet-off.hsPLA(2)) in HeLa cells. Providing additional tet-transactivator (tTA) protein either by stable transfection or coinfection with a tTA-expressing adenovirus resulted in a further increase of LacZ and sPLA(2) expression. Transgene expression in vitro was eliminated by the addition of tetracycline to the culture medium. Adtet-off.LacZ and Adtet-off.hsPLA(2) allowed successful gene transfer in vivo to lung and liver. While the expression was highly efficient within the lungs, however, additional tTA was necessary to achieve high-level expression within liver. CONCLUSIONS: Tet-regulatable adenoviral expression systems may facilitate the construction of recombinant adenoviruses encoding potentially toxic transgenes and permit regulated transgene expression.
Subject(s)
Adenoviridae/metabolism , Gene Transfer Techniques , Liver , Lung , Phospholipases A/genetics , Tetracycline/pharmacology , Transgenes , Adenoviridae/genetics , Animals , Cells, Cultured , Female , Gene Expression Regulation , Gene Targeting , Genetic Vectors , Group II Phospholipases A2 , HeLa Cells , Humans , Lac Operon/physiology , Liver/metabolism , Lung/metabolism , Mice , Mice, Inbred C57BL , Phospholipases A/metabolism , Recombinant Fusion Proteins/metabolism , Trans-Activators/physiologyABSTRACT
The nuclear bile acid receptor FXR has been proposed to play a central role in the feedback repression of the gene encoding cholesterol 7 alpha-hydroxylase (CYP7A1), the first and rate-limiting step in the biosynthesis of bile acids. We demonstrate that FXR directly regulates expression of fibroblast growth factor-19 (FGF-19), a secreted growth factor that signals through the FGFR4 cell-surface receptor tyrosine kinase. In turn, FGF-19 strongly suppresses expression of CYP7A1 in primary cultures of human hepatocytes and mouse liver through a c-Jun N-terminal kinase (JNK)-dependent pathway. This signaling cascade defines a novel mechanism for feedback repression of bile acid biosynthesis and underscores the vital role of FXR in the regulation of multiple pathways of cholesterol catabolism in the liver.
