Hepatic cholesterol 7-alpha-hydroxylase catalyzes the initial and rate- limiting step in the major pathway where by cholesterol is converted to bile salts. As such, 7-alpha-hydroxylase plays a central role both in the maintenance of whole body sterol balance and, secondarily, in the regulation of serum lipoprotein concentrations. Hepatic 7-alpha- hydroxylase is regulated at the transcriptional level in response to bile salts, fatty acids and, in some species, cholesterol; however, the molecular mechanisms where by these major physiological regulators alter transcription of the 7-alpha-hydroxylase gene are completely unknown. This is due, in large part, to the lack of in vitro or cell culture systems that reproduce the major forms of regulation observed in vivo. Moreover, transgenic rats containing promoter/reporter constructs up to 4 kb of 7-alpha-hydroxylase 5 -flanking DNA manifested very low levels of transgene expression that were minimally regulated by bile slats or cholesterol indicating that sequences other than those present in the 4 kb of 5'-flanking DNA are required for normal expression and regulation in vivo. The overall goal of the proposed research is to understand how transcription of the 7-alpha- hydroxylase gene is regulated by bile salts and fatty acids and to determine the mechanism whereby cholesterol up regulates transcription of the 7-alpha-hydroxylase gene in some species (rats and mice) but not in others (hamsters). Using nuclei and nuclear extracts from animals fed bile salts, bile salt sequestrants, fatty acids or cholesterol, we propose a systematic evaluation of the rat and hamster 7 -alpha-hydroxylase gene loci starting with DNase I hypersensitive site mapping to identify important regulatory sequences followed by DNase I footprinting and methylation interference studies to identify nuclear factor binding sites. Selected footprints will be analyzed using gel mobility shift assays and competitor oligonucleotides to identify known transcription factors. Potential enhances identified in this manner will be cloned next to the 7-alpha-hydroxylase proximal promoter (or a heterologous promoter) and testing for enhancer activity and the ability to confer responsiveness to physiologic regulators in vivo using adenovirus-mediated gene transfer. In the unlikely event that the sequences mediating responsiveness to the major physiological regulators cannot be identified using the in vivo transient transfection studies outline above, we will use transgenic animals to map the sequences responsible for regulation. Transgenic mice will initially be generated using P1 clone of the rat 7-alpha-hydroxylase gene. Deletional mutagenesis analysis will then be carried out guided by the results of DNase I hypersensitive site mapping. These studies will provide critical information regarding the transcriptional regulation of the 7-alpha-hydroxylase gene in vivo and may open the door for the development of novel strategies aimed at enhancing the conversion of cholesterol to bile salts and reducing cardiovascular risk.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL047551-05
Application #
2735196
Study Section
Metabolism Study Section (MET)
Project Start
1993-09-01
Project End
2001-06-30
Budget Start
1998-07-01
Budget End
1999-06-30
Support Year
5
Fiscal Year
1998
Total Cost
Indirect Cost
Name
University of Texas Sw Medical Center Dallas
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
City
Dallas
State
TX
Country
United States
Zip Code
75390
Vasandani, Chandna; Kafrouni, Abdallah I; Caronna, Antonella et al. (2002) Upregulation of hepatic LDL transport by n-3 fatty acids in LDL receptor knockout mice. J Lipid Res 43:772-84
Alam, K; Meidell, R S; Spady, D K (2001) Effect of up-regulating individual steps in the reverse cholesterol transport pathway on reverse cholesterol transport in normolipidemic mice. J Biol Chem 276:15641-9
Spady, D K; Willard, M N; Meidell, R S (2000) Role of acyl-coenzyme A:cholesterol acyltransferase-1 in the control of hepatic very low density lipoprotein secretion and low density lipoprotein receptor expression in the mouse and hamster. J Biol Chem 275:27005-12
Spady, D K; Kearney, D M; Hobbs, H H (1999) Polyunsaturated fatty acids up-regulate hepatic scavenger receptor B1 (SR-BI) expression and HDL cholesteryl ester uptake in the hamster. J Lipid Res 40:1384-94
Spady, D K; Cuthbert, J A; Willard, M N et al. (1998) Overexpression of cholesterol 7alpha-hydroxylase (CYP7A) in mice lacking the low density lipoprotein (LDL) receptor gene. LDL transport and plasma LDL concentrations are reduced. J Biol Chem 273:126-32
Woollett, L A; Spady, D K (1997) Kinetic parameters for high density lipoprotein apoprotein AI and cholesteryl ester transport in the hamster. J Clin Invest 99:1704-13
Woollett, L A; Kearney, D M; Spady, D K (1997) Diet modification alters plasma HDL cholesterol concentrations but not the transport of HDL cholesteryl esters to the liver in the hamster. J Lipid Res 38:2289-302
Spady, D K; Cuthbert, J A; Willard, M N et al. (1996) Feedback regulation of hepatic 7alpha-hydroxylase expression by bile salts in the hamster. J Biol Chem 271:18623-31
Feingold, K R; Spady, D K; Pollock, A S et al. (1996) Endotoxin, TNF, and IL-1 decrease cholesterol 7 alpha-hydroxylase mRNA levels and activity. J Lipid Res 37:223-8
Horton, J D; Cuthbert, J A; Spady, D K (1995) Regulation of hepatic 7 alpha-hydroxylase expression and response to dietary cholesterol in the rat and hamster. J Biol Chem 270:5381-7

Showing the most recent 10 out of 13 publications