Low-density Lipoprotein Receptor Messenger Research Articles

Hepatic tissue sterol regulatory element binding protein 2 and low-density lipoprotein receptor in nephrotic syndrome

Hypercholesterolemia is a main feature of nephrotic syndrome (NS) and is, in part, caused by acquired low-density lipoprotein (LDL) receptor deficiency. The LDL receptor deficiency in NS is accompanied by normal hepatic LDL receptor messenger RNA (mRNA) abundance. Expression of LDL receptor, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, and several other cholesterol-regulatory factors is regulated by sterol regulatory element binding protein 2 (SREBP-2). This study tested the hypothesis that nephrotic hypercholesterolemia may be associated with dysregulation of hepatic tissue SREBP-2 abundance or activity. Protein and mRNA abundance of SREBP-2, LDL receptor, and HMG-CoA reductase was determined in the livers of rats with chronic puromycin-induced NS and of control rats. The nephrotic group showed heavy proteinuria, hypoalbuminemia, severe hypercholesterolemia, and normal liver tissue total and free cholesterol concentrations. Despite severe hypercholesterolemia, the inactive microsomal and the active nuclear SREBP-2 levels were unchanged in the liver of the nephrotic animals. This was associated with a marked reduction in LDL receptor protein abundance. In confirmation of our earlier studies, LDL receptor and HMG-CoA reductase mRNA levels were unchanged in nephrotic animals. Hepatic SREBP-2 abundance and activity in hypercholesterolemic nephrotic rats were similar to those found in the normocholesterolemic control animals, representing a maladaptive response. This paradox may be, in part, due to acquired LDL receptor deficiency that helps sustain SREBP-2 expression/activity and maintain hypercholesterolemia by limiting hepatic cholesterol uptake. This is because SREBP-2 expression and activity are, in part, regulated by intracellular as opposed to plasma cholesterol.

Effects of chenodeoxycholic acid and deoxycholic acid on cholesterol absorption and metabolism in humans

Quantitative and qualitative differences in intralumenal bile acids may affect cholesterol absorption and metabolism. To test this hypothesis, 2 cross-over outpatient studies were conducted in adults with apo-A IV 1/1 or apo-E 3/3 genotypes. Study 1 included 11 subjects 24 to 37 years of age, taking 15 mg/kg/day chenodeoxycholic acid (CDCA) or no bile acid for 20 days while being fed a controlled diet. Study 2 included 9 adults 25 to 38 years of age, taking 15 mg/kg/day deoxycholic acid (DCA) or no bile acid, following the same experimental design and procedures as study 1. CDCA had no effect on plasma lipid concentrations, whereas DCA decreased (P < 0.05) plasma high-density lipoprotein (HDL)-cholesterol and tended to decrease (P = 0.15) low-density lipoprotein (LDL)-cholesterol. CDCA treatment enriched (P < 0.0001) bile with CDCA and increased cholesterol concentration in micelles, whereas meal-stimulated bile acid concentrations were decreased. DCA treatment enriched (P < 0.0001) bile with DCA and tended to increase intralumenal cholesterol solubilized in micelles (P = 0.06). No changes were found in cholesterol absorption, free cholesterol fractional synthetic rate (FSR), or 3-hydroxy-3 methylglutaryl (HMG) CoA reductase and LDL receptor messenger ribonucleic acid (mRNA) levels after CDCA treatment. DCA supplementation tended to decrease cholesterol absorption and reciprocally increase FSR and HMG CoA reductase and LDL receptor mRNA levels. Results of these 2 studies suggest that the solubilization of cholesterol in the intestinal micelles is not a rate-limiting step for its absorption.

Expression of low density lipoprotein receptor messenger ribonucleic acid in placentas from pregnancies with intrauterine growth retardation.

The low density lipoprotein receptor plays an important role in the uptake of maternal plasma lipoproteins for placental steroid metabolism. Using an RNase protection assay, the expression of low density lipoprotein receptor mRNA was measured in human placentas from pregnancies in the second and third trimester and from pregnancies with intrauterine growth retardation. Pregnancies complicated by intrauterine growth retardation showed a significantly higher expression (1.28), compared with the term and preterm pregnancies (P < 0.05). The increased low density lipoprotein receptor expression in placentas from pregnancies with intrauterine growth retardation could be a compensatory mechanism for the lower circulating low density lipoprotein concentrations in women with intrauterine growth retardation.

Concerted regulation of low density lipoprotein receptor gene expression by follicle-stimulating hormone and insulin-like growth factor I in porcine granulosa cells: promoter activation, messenger ribonucleic acid stability, and sterol feedback.

Insulin-like growth factor I (IGF-I) and the gonadotropin, FSH, can synergize to stimulate progesterone production in primary cultures of maturing granulosa cells. These trophic hormones increase low density lipoprotein (LDL) receptor binding and internalization, and the utilization of LDL-borne cholesterol by granulosa cells. To determine whether and how IGF-I and FSH control the genomic expression of the LDL receptor, we evaluated their individual and concerted effects on LDL receptor messenger RNA (mRNA) accumulation, stability, and gene promoter activity in first passage monolayer (serum-free) cultures of porcine granulosa cells. Ribonuclease protection assays revealed that LDL receptor mRNA accumulation was increased by human recombinant IGF-I (100 ng/ml), FSH (25 ng/ml NIDDK oFSH-20), or their combination by 2.2-, 2.6-, and 4.6-fold, respectively (P < 0.01). Hormonally stimulated LDL receptor mRNA accumulation was suppressed by 54-75% by the concurrent addition of LDL substrate (50 microg/ml). The combination of FSH and IGF-I significantly prolonged the message half-life, even in the presence of LDL. Using a combination of rapid amplification of cDNA 5'-ends, PCR with adapter-ligated genomic DNA, Southern hybridization, and DNA sequencing, we isolated 1076 bp of the porcine LDL receptor gene upstream of the coding region. In transient transfection assays, with a pLDLR1076/luciferase plasmid construct, FSH, FSH plus IGF-I, or 8-bromo-cAMP (1 mM) treatment (but not IGF-I alone) increased luciferase reporter gene activity by 10- to 23-fold in porcine granulosa cells. Over time in serum-free culture, the basal activity of the LDL receptor gene promoter increased and eventually surpassed hormone-stimulated effects, but was suppressed by LDL substrate (by 75%) at 24 h. The foregoing stimulatory hormone effects and sterol repression were localized to a 116-bp region in the porcine promoter between -255 and -139 upstream of the translational start site. We conclude that the combination of FSH and IGF-I can induce accumulation of LDL receptor mRNA in cultured granulosa cells even in the presence of sterol negative feedback and can do so mechanistically by a combination of promoter activation and increased mRNA stability.

Concerted Regulation of Low Density Lipoprotein Receptor Gene Expression by Follicle-Stimulating Hormone and Insulin-Like Growth Factor I in Porcine Granulosa Cells: Promoter Activation, Messenger Ribonucleic Acid Stability, and Sterol Feedback

Insulin-like growth factor I (IGF-I) and the gonadotropin, FSH, can synergize to stimulate progesterone production in primary cultures of maturing granulosa cells. These trophic hormones increase low density lipoprotein (LDL) receptor binding and internalization, and the utilization of LDL-borne cholesterol by granulosa cells. To determine whether and how IGF-I and FSH control the genomic expression of the LDL receptor, we evaluated their individual and concerted effects on LDL receptor messenger RNA (mRNA) accumulation, stability, and gene promoter activity in first passage monolayer (serum-free) cultures of porcine granulosa cells. Ribonuclease protection assays revealed that LDL receptor mRNA accumulation was increased by human recombinant IGF-I (100 ng/ml), FSH (25 ng/ml NIDDK oFSH-20), or their combination by 2.2-, 2.6-, and 4.6-fold, respectively (P < 0.01). Hormonally stimulated LDL receptor mRNA accumulation was suppressed by 54–75% by the concurrent addition of LDL substrate (50 μg/ml). The combination of FSH and IGF-I significantly prolonged the message half-life, even in the presence of LDL. Using a combination of rapid amplification of cDNA 5′-ends, PCR with adapter-ligated genomic DNA, Southern hybridization, and DNA sequencing, we isolated 1076 bp of the porcine LDL receptor gene upstream of the coding region. In transient transfection assays, with a pLDLR1076/luciferase plasmid construct, FSH, FSH plus IGF-I, or 8-bromo-cAMP (1 mm) treatment (but not IGF-I alone) increased luciferase reporter gene activity by 10- to 23-fold in porcine granulosa cells. Over time in serum-free culture, the basal activity of the LDL receptor gene promoter increased and eventually surpassed hormone-stimulated effects, but was suppressed by LDL substrate (by 75%) at 24 h. The foregoing stimulatory hormone effects and sterol repression were localized to a 116-bp region in the porcine promoter between −255 and −139 upstream of the translational start site. We conclude that the combination of FSH and IGF-I can induce accumulation of LDL receptor mRNA in cultured granulosa cells even in the presence of sterol negative feedback and can do so mechanistically by a combination of promoter activation and increased mRNA stability.

Dissociation of apolipoprotein and apolipoprotein receptor response to lesion in the rat brain: An in situ hybridization study

The ɛ4 allele of apolipoprotein E is associated with increased risk for developing Alzheimer's disease. To further understand the anatomical distribution of apolipoprotein E and its native receptors in the brain, we studied their messenger RNA expression in the adult rat brain under normal conditions and in response to an excitotoxic lesion to the hippocampus. In situ hybridization using oligonucleotide probes for apolipoprotein E, apolipoprotein J, the low density lipoprotein receptor, very low density lipoprotein receptor, low density lipoprotein receptor related protein, 39,000 mol. wt receptor-associated protein and glycoprotein 330/Megalin messenger RNA were performed on adjacent sections throughout the rat forebrain. Apolipoprotein E messenger RNA was abundantly expressed in the rat brain in both white and gray matter localizing to astrocytes but not neurons. Low density lipoprotein receptor-related protein and receptor-associated protein messenger RNA had a similar regional distribution but low density lipoprotein receptor-related protein messenger RNA was expressed by both neurons and glia, while the expression of receptor-associated protein messenger RNA was more highly expressed in neurons. Apolipoprotein J messenger RNA was expressed by neurons, glia and choroid plexus. The low density lipoprotein receptor and very low density lipoprotein receptor messenger RNA were found in both neurons and glia. Glycoprotein 330/Megalin messenger RNA was not detectable in the adult rat brain. In response to hippocampal lesions, apolipoprotein E and apolipoprotein J messenger RNAs were significantly upregulated seven and 11 days post-lesion but the expression of low density lipoprotein receptor, low density lipoprotein receptor-related protein, receptor-associated protein, glycoprotein 330/Megalin, and very low density lipoprotein receptor messenger RNAs were unchanged. The expression of apolipoprotein E messenger RNA increased gradually beginning at three days while the expression of apolipoprotein J messenger RNA began to increase at seven days post-lesion. These findings further implicate apolipoproteins in the response of the brain to injury in vivo and suggest that transcriptional up-regulation of the apolipoprotein receptors studied is not a prominent feature in the response.

Novel effects of histamine on lipoprotein metabolism: suppression of hepatic low density lipoprotein receptor expression and reduction of plasma high density lipoprotein cholesterol in the rat.

Histamine has been shown to be involved in atherosclerosis and coronary heart disease. Little information is available regarding the effects of histamine on lipoprotein metabolism. In the current study, we investigated the effects of histamine on the expression of hepatic low density lipoprotein (LDL) receptors and on plasma lipoproteins in the rat. Injection of compound 48/80 (C48/80, a histamine releaser) or histamine reduced hepatic LDL receptor expression, but not LDL receptor messenger RNA levels. Oral administration of polymyxin B (an antiendotoxin antibiotic and a histamine releaser) before the injection of C48/80 or histamine did not attenuate their effects. Polymyxin B itself had effects similar to those of C48/80 and histamine on LDL receptors. These results suggest that the effects of histamine are not mediated by the induction of gut-derived endotoxemia. Histamine H2 agonists (dimaprit and impromidine), but not H1 agonists (2-methylhistamine and 2-thiazolylethylamine), also reduced hepatic LDL receptor expression. The suppressive effect of C48/80 on hepatic LDL receptor expression was not attenuated by either the H1 antagonist (chlorpheniramine) or the H2 antagonist (cimetidine). Administration of C48/80 also reduced plasma high density lipoprotein (HDL) cholesterol. The H1 antagonist (chlorpheniramine), but not the H2 antagonist (cimetidine), almost completely reversed the effect of C48/80 on plasma HDL cholesterol. In conclusion, histamine suppresses hepatic LDL receptor expression via a non-H1 receptor-mediated pathway, and histamine reduces plasma HDL cholesterol via an H1 receptor-mediated pathway.

Decreased very-low-density lipoprotein and low-density lipoprotein receptor messenger ribonucleic acid expression in placentas from preeclamptic pregnancies: Murata M.; Kodama H.; Goto K.; Hirano H.; Tanaka T. Japan Am J Obstet Gynecol 1996 175/6 (1551–1556)

Decreased very-low-density lipoprotein and low-density lipoprotein receptor messenger ribonucleic acid expression in placentas from preeclamptic pregnancies: Murata M.; Kodama H.; Goto K.; Hirano H.; Tanaka T. Japan Am J Obstet Gynecol 1996 175/6 (1551–1556)

Decreased very-low-density lipoprotein and low-density lipoprotein receptor messenger ribonucleic acid expression in placentas from preeclamptic pregnancies

OBJECTIVE: Our purpose was to investigate the levels of very-low-density lipoprotein and low-density lipoprotein receptor messenger ribonucleic acid expression in placentas obtained from normal pregnant women at various gestational stages and from patients with preeclampsia. STUDY DESIGN: The relative level of expression of very-low-density lipoprotein and low-density lipoprotein receptor messenger ribonucleic acid in each sample was determined by Northern blot analysis as a ratio of the intensity to that of glyceraldehyde-3-phosphate dehydrogenase messenger ribonucleic acid. RESULTS: The expression of very-low-density lipoprotein receptor messenger ribonucleic acid increased significantly ( p < 0.05) from both the first ( n = 9) and second ( n = 8) trimesters to the third ( n = 11) trimester. Similarly, the expression of low-density lipoprotein receptor messenger ribonucleic acid increased significantly ( p < 0.05) from the first to the third trimester. The expression of both very-low-density lipoprotein and low-density lipoprotein receptor messenger ribonucleic acids in third-trimester placentas from preeclamptic pregnancies ( n = 13) was significantly lower than that in normal pregnancies in the third trimester. CONCLUSION: These results demonstrate the expression of very-low-density lipoprotein and low-density lipoprotein receptor messenger ribonucleic acids in the human placenta, both of which increase at late gestational stages, and the decreased expression in cases of preeclampsia. Abnormal fetomaternal lipid metabolism resulting from low expression of these receptors in the placenta may be involved in the pathogenesis of preeclampsia. (Am J Obstet Gynecol 1996;175:1551-6.)

Cyclosporine inhibits catabolism of low-density lipoproteins in HepG2 cells by about 25%

The aim of this study was to elucidate the possible causes of elevated low-density lipoprotein (LDL)-cholesterol levels in transplanted patients treated with the immunosuppressant drug, cyclosporine. HepG2 cells, from a well-differentiated cell-line of hepatoma cells, were cultured and used as a model for in vitro hepatocytic LDL uptake. Different concentrations of cyclosporine, which were within the range of concentrations found in humans treated with cyclosporine, were added to tissue culture medium together with 125I-LDL. The results showed that cyclosporine reduced LDL uptake and degradation in HepG2 cells by about 25%. The cells were also pretreated with cyclosporine for 1 to 24 hours and then incubated with new medium containing labeled LDL for 2 hours at 4 degrees C in an LDL-binding assay. The data showed that cyclosporine reduced the subsequent LDL binding. Cyclosporine has no toxic effects on HepG2 cells, as shown by unchanged growth capacity of the cells. By means of a 50-fold excess of unlabeled LDL, a monoclonal anti-LDL receptor antibody, and dextran sulfate, we also evaluated if this inhibition of LDL binding occurred through the LDL receptor- mediated pathway, through non-LDL receptor-mediated pathways, or through both. The results show that cyclosporine reduces LDL binding and uptake by mainly inhibiting the LDL receptor-mediated pathway. We also studied the effect of the LDL-cyclosporine complex on the binding of labelled LDL. The presence of cyclosporine in the LDL particle does not influence the binding behaviour of LDL to its receptor. We also found that cyclosporine reduces the expression of the LDL receptor messenger RNA (mRNA) by about 40%. Thus, the interpretation of this study is that cyclosporine can cause an increase in LDL-cholesterol in the plasma of transplantation patients by reducing the catabolism of LDL in the liver by inhibiting mainly the LDL receptor-mediated catabolism through an effect on LDL receptor synthesis. (Hepatology 1996 Sep;24(3):613-9)

Cyclosporine inhibits catabolism of low-density lipoproteins in HepG2 cells by about 25%

The aim of this study was to elucidate the possible causes of elevated low-density lipoprotein (LDL)-cholesterol levels in transplanted patients treated with the immunosuppressant drug, cyclosporine. HepG2 cells, from a well-differentiated cell-line of hepatoma cells, were cultured and used as a model for in vitro hepatocytic LDL uptake. Different concentrations of cyclosporine, which were within the range of concentrations found in humans treated with cyclosporine, were added to tissue culture medium together with 125I-LDL. The results showed that cyclosporine reduced LDL uptake and degradation in HepG2 cells by about 25%. The cells were also pretreated with cyclosporine for 1 to 24 hours and then incubated with new medium containing labeled LDL for 2 hours at 4 degrees C in an LDL-binding assay. The data showed that cyclosporine reduced the subsequent LDL binding. Cyclosporine has no toxic effects on HepG2 cells, as shown by unchanged growth capacity of the cells. By means of a 50-fold excess of unlabeled LDL, a monoclonal anti-LDL receptor antibody, and dextran sulfate, we also evaluated if this inhibition of LDL binding occurred through the LDL receptor-mediated pathway, through non-LDL receptor-mediated pathways, or through both. The results show that cyclosporine reduces LDL binding and uptake by mainly inhibiting the LDL receptor-mediated pathway. We also studied the effect of the LDL-cyclosporine complex on the binding of labelled LDL. The presence of cyclosporine in the LDL particle does not influence the binding behaviour of LDL to its receptor. We also found that cyclosporine reduces the expression of the LDL receptor messenger RNA (mRNA) by about 40%. Thus, the interpretation of this study is that cyclosporine can cause an increase in LDL-cholesterol in the plasma of transplantation patients by reducing the catabolism of LDL in the liver by inhibiting mainly the LDL receptor-mediated catabolism through an effect on LDL receptor synthesis.

Differences in the mechanisms of uptake and endocytosis of small and large chylomicron remnants by rat liver

Initial binding and subsequent endocytosis of small and large chylomicron remnants by rat liver were compared. Small and large chylomicrons were obtained from mesenteric lymph of glucose- or fat-fed rats, respectively. The low-density lipoprotein (LDL) receptor was up- and down-regulated as shown by LDL receptor messenger RNA (mRNA). The rate of removal of small chylomicron remnants by isolated perfused rat livers followed closely the activity of the LDL receptor. When mRNA was undetectable, the uptake was as low as that of lymphatic small chylomicrons. In contrast, the uptake of large chylomicron remnants into perfused rat livers was unaffected by changes of the LDL-receptor activity, but significantly reduced after livers were flushed with heparin or heparinase. Large chylomicron remnants were cleared from plasma much faster than small chylomicron remnants, but were more slowly internalized into hepatocytes. Both, small and large chylomicron remnants entered the pathway of receptor-mediated endocytosis as shown by electron microscopy and analysis of isolated endosomes. Yet, large chylomicron remnants were taken up into the compartment of uncoupling of receptors and ligands and multivesicular bodies at a much slower rate. This was independent of the activity of the LDL receptor and the heparin-releasable binding site. From these findings it is concluded that large chylomicron remnants initially bind rapidly to surface components other than the LDL receptor, one of which may be hepatic lipase. Yet, the consecutive internalization is slow. In contrast, small chylomicron remnants are removed at a slower rate from plasma, binding predominantly to the LDL receptor, but are more readily taken up into endosomes. (Hepatology 1996 Aug;24(2):344-51)

Developmental increase in low density lipoprotein receptor messenger ribonucleic acid levels in placental syncytiotrophoblasts during baboon pregnancy

We have previously shown that there was an estrogen-regulated developmental increase in low density lipoprotein (LDL) uptake by placental syncytiotrophoblasts during baboon pregnancy. To determine whether this reflected enhanced expression of the LDL receptor, the levels of LDL receptor messenger RNA (mRNA) were determined by Northern blot and reverse transcription-polymerase chain reaction in placental tissue obtained from baboons (Papio anubis) in early (days 58-64; pooled to yield 5 samples), mid- (days 97-110; pooled to yield 12 samples), and late (days 161-175; pooled to yield 15 samples) gestation (term = 184 days). Whole villous tissue and a trophoblast cell fraction isolated by 50% Percoll gradient centrifugation were analyzed. The latter cell fraction was equally comprised predominantly of syncytiotrophoblasts at early, mid-, and late gestation as determined by extensive immunocytochemical reactivity with antisera to syncytiotrophoblast-specific peptides. Tissues were extracted with guanidine isothiocyanate and 5 micrograms polyadenylated-enriched RNA hybridized to a 32P-labeled human LDL receptor complementary DNA (cDNA). A major 6.2-kilobase LDL receptor mRNA transcript was expressed in syncytiotrophoblasts and whole villous tissue, as determined by Northern blot. In the syncytiotrophoblast-rich cell fraction, LDL receptor mRNA levels, analyzed by Northern blot and autoradiodensitometry and expressed as a ratio of beta-actin, were similarly low in early (0.66 +/- 0.12 arbitrary units) and mid- (1.15 +/- 0.23) gestation, then increased to a level in late gestation (2.71 +/- 0.33) that was over 4- and 2-fold greater (P < 0.01) than that in early or midgestation, respectively. In contrast, in whole villous tissue, LDL receptor and beta-actin mRNA levels exhibited no consistent change or decreased slightly with advancing pregnancy, so that when corrected for beta-actin, LDL receptor mRNA levels were similar in early (1.53 +/- 0.33), mid- (1.44 +/- 0.16), and late (2.32 +/- 0.29) gestation. The unchanged levels of LDL receptor mRNA in whole placental villous tissue with advancing primate gestation may reflect potential villous tissue with advancing primate gestation may reflect potential confounding effects that nontrophoblast, e.g. vascular, components of the developing placenta may have on assessing trophoblast endocrine function in whole villous tissue. Amplification of trophoblast RNA by reverse transcription-polymerase chain reaction with LDL receptor primers generated a single cDNA product of approximately 258 base pairs.

Developmental increase in low density lipoprotein receptor messenger ribonucleic acid levels in placental syncytiotrophoblasts during baboon pregnancy.

We have previously shown that there was an estrogen-regulated developmental increase in low density lipoprotein (LDL) uptake by placental syncytiotrophoblasts during baboon pregnancy. To determine whether this reflected enhanced expression of the LDL receptor, the levels of LDL receptor messenger RNA (mRNA) were determined by Northern blot and reverse transcription-polymerase chain reaction in placental tissue obtained from baboons (Papio anubis) in early (days 58-64; pooled to yield 5 samples), mid- (days 97-110; pooled to yield 12 samples), and late (days 161-175; pooled to yield 15 samples) gestation (term = 184 days). Whole villous tissue and a trophoblast cell fraction isolated by 50% Percoll gradient centrifugation were analyzed. The latter cell fraction was equally comprised predominantly of syncytiotrophoblasts at early, mid-, and late gestation as determined by extensive immunocytochemical reactivity with antisera to syncytiotrophoblast-specific peptides. Tissues were extracted with guanidine isothiocyanate and 5 micrograms polyadenylated-enriched RNA hybridized to a 32P-labeled human LDL receptor complementary DNA (cDNA). A major 6.2-kilobase LDL receptor mRNA transcript was expressed in syncytiotrophoblasts and whole villous tissue, as determined by Northern blot. In the syncytiotrophoblast-rich cell fraction, LDL receptor mRNA levels, analyzed by Northern blot and autoradiodensitometry and expressed as a ratio of beta-actin, were similarly low in early (0.66 +/- 0.12 arbitrary units) and mid- (1.15 +/- 0.23) gestation, then increased to a level in late gestation (2.71 +/- 0.33) that was over 4- and 2-fold greater (P < 0.01) than that in early or midgestation, respectively. In contrast, in whole villous tissue, LDL receptor and beta-actin mRNA levels exhibited no consistent change or decreased slightly with advancing pregnancy, so that when corrected for beta-actin, LDL receptor mRNA levels were similar in early (1.53 +/- 0.33), mid- (1.44 +/- 0.16), and late (2.32 +/- 0.29) gestation. The unchanged levels of LDL receptor mRNA in whole placental villous tissue with advancing primate gestation may reflect potential villous tissue with advancing primate gestation may reflect potential confounding effects that nontrophoblast, e.g. vascular, components of the developing placenta may have on assessing trophoblast endocrine function in whole villous tissue. Amplification of trophoblast RNA by reverse transcription-polymerase chain reaction with LDL receptor primers generated a single cDNA product of approximately 258 base pairs.(ABSTRACT TRUNCATED AT 400 WORDS)

Growth hormone specifically stimulates the expression of low density lipoprotein receptors in human hepatoma cells

Human GH (hGH) has been shown to stimulate hepatic low density lipoprotein (LDL) receptor expression in man in vivo. To further characterize this effect in vitro, we determined the expression of LDL receptors in cultured human hepatoma (HepG2) cells exposed to hGH. After incubation with hGH, stimulation of LDL receptors appeared at a concentration of 0.25 nM hGH. The presence of hGH receptors on HepG2 cells could be demonstrated by immunocytochemistry using a hGH receptor-specific monoclonal antibody. Binding studies, using 125I-labeled hGH, revealed high affinity binding with the appropriate somatogenic specificity. The LDL receptor induction was specific for hGH, as both bovine GH and recombinant human PRL were without effect. The LDL receptor stimulation occurred in parallel with increased levels of LDL receptor messenger RNA. Inclusion of dexamethasone and thyroid hormone in the incubation medium enhanced the LDL receptor stimulation by hGH. Although incubation with insulin-like growth factor-I (IGF-I) stimulated LDL receptor expression, the hGH-induced stimulation was unaltered after preincubation of cells with a monoclonal mouse anti-IGF-I antibody, suggesting that the release of IGF-I is not involved in LDL receptor stimulation by hGH. We conclude that hGH specifically induces the LDL receptor in cultured HepG2 cells at both the protein and the messenger RNA level, and that the induction is independent of IGF-I release.

Growth hormone specifically stimulates the expression of low density lipoprotein receptors in human hepatoma cells.

Human GH (hGH) has been shown to stimulate hepatic low density lipoprotein (LDL) receptor expression in man in vivo. To further characterize this effect in vitro, we determined the expression of LDL receptors in cultured human hepatoma (HepG2) cells exposed to hGH. After incubation with hGH, stimulation of LDL receptors appeared at a concentration of 0.25 nM hGH. The presence of hGH receptors on HepG2 cells could be demonstrated by immunocytochemistry using a hGH receptor-specific monoclonal antibody. Binding studies, using 125I-labeled hGH, revealed high affinity binding with the appropriate somatogenic specificity. The LDL receptor induction was specific for hGH, as both bovine GH and recombinant human PRL were without effect. The LDL receptor stimulation occurred in parallel with increased levels of LDL receptor messenger RNA. Inclusion of dexamethasone and thyroid hormone in the incubation medium enhanced the LDL receptor stimulation by hGH. Although incubation with insulin-like growth factor-I (IGF-I) stimulated LDL receptor expression, the hGH-induced stimulation was unaltered after preincubation of cells with a monoclonal mouse anti-IGF-I antibody, suggesting that the release of IGF-I is not involved in LDL receptor stimulation by hGH. We conclude that hGH specifically induces the LDL receptor in cultured HepG2 cells at both the protein and the messenger RNA level, and that the induction is independent of IGF-I release.

Decreased low-density lipoprotein receptor function and mRNA levels in lymphocytes from uremic patients

The mechanisms by which renal failure causes hyperlipoproteinemia remain unclear. To investigate the potential role of the low-density lipoprotein (LDL) receptor in lipoprotein metabolism in uremia we measured LDL receptor function in peripheral blood mononuclear cells (PBMC) from uremic patients and control subjects using a functional assay in which proliferation of lectin-stimulated PBMC in the presence of lovastatin was dependent upon internalization of exogenous cholesterol via a functional LDL receptor. The amount of LDL required to reverse 50% of lovastatin-induced inhibition of proliferation in PBMC from uremic patients was significantly greater (3.6 +/- 1.8 micrograms/ml, N = 33, P < 0.05) than controls, (1.99 +/- 0.6 micrograms/ml, N = 37). Abnormal LDL receptor function in four uremic patients normalized following renal transplantation. To investigate the molecular basis for LDL receptor dysfunction, we directly quantitated LDL receptor messenger RNA (mRNA) in PBMC from uremic patients and control subjects using a ribonuclease protection assay. LDL receptor mRNA expression in uremic patients was 0.42 +/- 0.08 (N = 10), significantly lower (P < 0.015) than in normal subjects, 0.71 +/- 0.08 (N = 14). These data suggest that an acquired defect in LDL receptor function in PBMC from uremic patients exists which may be due to decreased LDL receptor expression. These abnormalities, if present in other tissues, could contribute to the aberrant lipoprotein metabolism which is a consistent feature of uremia.

Gene expression in macrophage-rich human atherosclerotic lesions. 15-lipoxygenase and acetyl low density lipoprotein receptor messenger RNA colocalize with oxidation specific lipid-protein adducts.

Oxidatively modified low density lipoprotein (LDL) exhibits several potentially atherogenic properties, and inhibition of LDL oxidation in rabbits decreases the rate of the development of atherosclerotic lesions. In vitro studies have suggested that cellular lipoxygenases may be involved in LDL oxidation, and we have shown previously that 15-lipoxygenase and oxidized LDL are present in rabbit atherosclerotic lesions. We now report that epitopes of oxidized LDL are also found in macrophage-rich areas of human fatty streaks as well as in more advanced human atherosclerotic lesions. Using in situ hybridization and immunostaining techniques, we also report that 15-lipoxygenase mRNA and protein colocalize to the same macrophage-rich areas. Moreover, these same lesions express abundant mRNA for the acetyl LDL receptor but no detectable mRNA for the LDL receptor. We suggest that atherogenesis in human arteries may be linked to macrophage-induced oxidative modification of LDL mediated by 15-lipoxygenase, leading to subsequent enhanced macrophage uptake, partly by way of the acetyl LDL receptor.

Thyrotropin modulates low density lipoprotein binding activity in FRTL-5 thyroid cells.

FRTL-5 cells possess high affinity low density lipoprotein (LDL) receptors which bind, internalize, and degrade LDL. When FRTL-5 cells are deprived of thyrotropin (TSH) the binding of LDL increases more than 2-fold. Upon addition of TSH, at a concentration of 1 x 10(-10) M or greater, LDL binding decreases rapidly and within 24 h reaches the level which is typical of FRTL-5 cells chronically stimulated by TSH. The data available suggest that TSH-dependent down-regulation of LDL receptor activity is exerted through a reduction of the number of active LDL receptors, with no change in affinity. It is unlikely that the synthesis of LDL receptors is impaired, since LDL receptor messenger RNA is not decreased by TSH. The effect of the hormone on LDL receptor activity can be mimicked by 8-Br-cAMP and is completely abolished by the protein synthesis inhibitor cycloheximide but not by actinomycin D. TSH regulation of LDL receptor activity is lost in v-ras Ki-transformed FRTL-5 cells (Ki Mol) which also have lost TSH dependence for adenylate cyclase activation and growth. However, 8-Br-cAMP decreases LDL binding in Ki Mol FRTL-5 cells. The reduced availability of LDL receptor in TSH-stimulated FRTL-5 cells may be related to the increased membrane fluidity (Beguinot, F., Beguinot, L., Tramontano, D., Duilio, C., Formisano, S., Bifulco, M., Ambesi-Impiombato, F. S., and Aloj, S. M. (1987) J. Biol. Chem. 262, 1575-1582) or may reflect increased degradation of LDL receptors. We propose that a lower cholesterol uptake is needed in an actively proliferating cell population, to increase the production of isoprenoids whether it be for cholesterol biosynthesis or for the synthesis of other compounds requiring isoprenoid precursors.