Low density lipoprotein receptor and 3-hydroxy-3-methylglutaryl coenzyme A reductase gene expression in human mononuclear leukocytes is regulated coordinately and parallels gene expression in human liver (original) (raw)
Related papers
The Journal of Clinical Endocrinology & Metabolism, 2002
To characterize the coordinate regulation of cholesterol metabolism in human liver, we simultaneously quantified mRNA levels of cholesterol 7␣-hydroxylase (CYP7A1), 3-hydroxy-3methylglutaryl coenzyme A reductase (HMGR), and lowdensity lipoprotein receptors (LDLRs) in liver biopsies from 76 patients undergoing cholecystectomy. The three transcript levels were not different between untreated gallstone and gallstone-free patients and not significantly altered by 10-d exclusion of dietary cholesterol. Treatment with chenodeoxycholic acid suppressed CYP7A1 and to a lesser extent HMGR mRNA levels. Cholestyramine treatment increased CYP7A1, but also HMGR and LDLR mRNA, and statins only increased HMGR mRNA. Resin ؉ statin treatment increased all mRNA species. In untreated patients, the mRNA levels of HMGR and LDLR were more strongly correlated (r ؍ ؉0.60) than those of CYP7A1 and HMGR (r ؍ ؉0.49) or CYP7A1 and LDLR (r ؍ ؉0.21). In the treated patients, in whom bile acid synthesis was suppressed or stimulated, mRNA levels of CYP7A1 and HMGR (r ؍ ؉0.84) as well as CYP7A1 and LDLR (r ؍ ؉0.62) were more strongly correlated than those of HMGR and LDLR (r ؍ ؉0.59). The coordinate control of HMGR and LDLR mRNA levels reflects their common regulation by shared transcriptional activation. In contrast, following changes in bile acid flux through the liver, CYP7A1 gene expression becomes a strong modulator of hepatic cholesterol metabolism.
The Journal of Biological Chemistry, 1991
Feeding rats diets containing 2% cholesterol markedly reduced hepatic 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase activity but had little effect on mRNA levels. Addition of mevalonolactone to the diet further decreased reductase activity independent of a change in mRNA levels. In contrast, farnesyl pyrophosphate synthetase mRNA levels and enzyme activity were decreased to similar degrees in response to dietary cholesterol. Addition of mevalonolactone to the diet did not further decrease farnesyl pyrophosphate synthetase activity. Dietary cholesterol and mevalonolactone had no effect on mRNA levels for "cellular nucleic acid-binding protein" which has been demonstrated to bind the sterol regulatory elements in the HMG-CoA reductase and farnesyl pyrophosphate synthetase promoters. Dietary cholesterol increased cholesterol 7a-hydroxylase mRNA levels as expected. These results suggest that cholesterol-mediated feedback regulation of hepatic HMG-CoA reductase gene expression does not occur at the level of transcription.
Journal of Biological Chemistry
Feeding rats diets containing 2% cholesterol markedly reduced hepatic 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase activity but had little effect on mRNA levels. Addition of mevalonolactone to the diet further decreased reductase activity independent of a change in mRNA levels. In contrast, farnesyl pyrophosphate synthetase mRNA levels and enzyme activity were decreased to similar degrees in response to dietary cholesterol. Addition of mevalonolactone to the diet did not further decrease farnesyl pyrophosphate synthetase activity. Dietary cholesterol and mevalonolactone had no effect on mRNA levels for "cellular nucleic acid-binding protein" which has been demonstrated to bind the sterol regulatory elements in the HMG-CoA reductase and farnesyl pyrophosphate synthetase promoters. Dietary cholesterol increased cholesterol 7 alpha-hydroxylase mRNA levels as expected. These results suggest that cholesterol-mediated feed-back regulation of hepatic HMG-CoA reductase gene expr...
Archives of Biochemistry and Biophysics, 1997
noblotting; transcription; cholesterol; lovastatin; sterol response element binding proteins. The mechanism by which dietary cholesterol regulates expression of the hepatic low-density lipoprotein (LDL) receptor was investigated. In a previous study (Arch. Biochem. Biophys. 325, 242-248, 1996), we dem-Regulation of low-density lipoprotein (LDL) 2 receptor onstrated that dietary cholesterol reduces the rate of gene expression by cholesterol is thought to occur ex-LDL receptor protein degradation without affecting clusively at the transcriptional level (1). The LDL resteady-state levels of receptor protein. In view of these findings, it was expected that dietary cholesterol ceptor provides cholesterol to cells by binding and interwould decrease the rate of transcription of the hepatic nalizing LDL. When cholesterol demands are high, the LDL receptor gene, resulting in lower mRNA levels LDL receptor gene is transcribed actively, and conseand lower rates of synthesis of LDL receptor protein. quently more LDL is internalized. However, when cho-Surprisingly, neither the rate of transcription nor the lesterol accumulates within the cell, transcription of level of LDL receptor mRNA was reduced in response this gene is repressed causing a decrease in LDL interto dietary cholesterol, even though hepatic cholesterol nalization. levels were increased twofold. This suggests that un-
Journal of Lipid Research, 1995
We designed a rapid method for determining mRNA content of cholesterol biosynthesis enzymes and LDL receptor (LDLR) using a ribonuclease protection assay (RPA). 32P-labeled cRNA fi-agments for genes of human LDLR and the enzymes HMGCoA synthase (HMGS), HMG-CoA reductase. (HMGR), mevalonate kinase (MK), farnesyl pyrophosphate synthase (FPPS), and squalene synthase (SQS) were prepared by in vitro transcription. Total RNA prepared from HepG2 cells was hybridized with the cRNA probe and the hybridized mRNA was determined under protection from RNase digestion. Probe content used in this assay was excess in determining the desired mRNA in total RNA, and surplus probes were completely digested using RNase under standard conditions. When cells were cultured in DMEM supplemented with 10% fetal calf serum (FCS), mRNA levels of FPPS, SQS, and LDLR were about 4to 7-fold higher than those of HMGS, HMGR, and MK. On incubation with DMEM supplemented with 10% lipoprotein-deficient serum (LPDS) for 8 h, all messenger RNA levels increased 1.5-to 3.5-fold. In addition, when the HMG-CoA reductase inhibitor compactin was added to 10% LPDS-DMEM, these levels increased even further and the change in mRNA level seemed to differ between the enzymes and LDLR. From these results, we conclude that RPA is a useful method for determining the very small amount of mRNA level of cholesterol biosynthesis enzymes and LDLR in the cell.Shimokawa, T., Y. Kawabe, M. Honda, Y. Yazaki, A. Matsumoto, H. Itakura, and T. Kodama. Determination of mRNA levels of cholesterol biosynthesis enzymes and LDL receptor using ribonuclease protection assay.
Clinical Chemistry
We have improved the protocol for RNA quantification by using RNase protection. Instead of precipitation and extraction with phenol and chloroform, we use a faster and more reliable precipitation based on guanidinium thiocyanate (GdSCN). The internal standard is produced by in vitro transcription of a DNA template constructed so as to allow simultaneous detection of the in vitro transcript and the low-density lipoprotein receptor (LDLR) mRNA by use of the same probe and hybridization. Addition of this internal standard at the step for RNA isolation reduced the analytical imprecision from 40.8% to 19.3%. Estimates of the within- and between-subject biological variations of the LDLR mRNA content in peripheral blood mononuclear cells (PBMCs) isolated from healthy volunteers were 21.5% and 13.6%, respectively, and the analytical imprecision was 22.6%. The mean content of LDLR mRNA in PBMCs from healthy individuals was 0.78 copies per cell.
Coordinate Regulation of Cholesterol 7α-Hydroxylase and HMG-CoA Reductase in the Liver
Subcellular Biochemistry, 1997
Cholesterol homeostasis in the liver is dependent upon the activity of a number of key structures: 3-hydroxy-3-methylglutaryl-CoA, HMG-CoA reductase (HMGR), acy 1 cholesterol acyltransferase, cholesterol 7 a-hydroxylase, and the LDL receptor (for a review, see Goldstein and Brown, 1990). The regulation of all these proteins is steroid dependent. This steroid-dependent regulation has been characterized in some detail at the gene level for HMG CoA synthase and the LDL receptor, and involves sophisticated coordinate transcriptional control, with interaction of transacting factors with sterol regulatory elements (SRE-l) sequences present in the promoter regions of these genes. In addition to this transcriptional control, there may also be regulatory control at different posttranscriptionallevels. The detailed mechanism behind the sterol-dependent regulation of the other key enzymes is not as well characterized at present.
Blood, 1994
Altered cholesterol homeostasis has been noted in malignant cells, which led us to explore the regulation of cholesterol metabolism in normal and leukemic cells. The mean low-density lipoprotein (LDL) receptor and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activities were fivefold and threefold higher in mononuclear blood cells from 33 patients with leukemia, compared with cells from 23 healthy subjects, whereas elevations in RNA levels were twofold and 40% only. The activities of the two proteins correlated in normal cells (r = .46), whereas an inverse correlation was found in leukemic cells (r = -.40). Relatively weak correlations were found between LDL receptor RNA levels and receptor activity in normal (r = .48) and leukemic cells (r = .49), and HMG-CoA reductase RNA levels correlated (r = .53) with reductase activity in leukemic cells only. The ratios of protein activities to RNA levels in cells were constant during consecutive blood samplings and similar in leuk...