Lipoprotein Receptor Gene Expression Research Articles

Impact of Bariatric Surgery on Subtilisin/Kexin Type 9 (PCSK9) Gene Expression and Inflammation in the Adipose Tissue of Obese Diabetic Rats.

Bariatric surgery improves dyslipidaemia and reduces body weight, but it remains unclear how bariatric surgery modulates gene expression in fat cells to influence the proprotein convertase subtilisin/kexin type 9 (PCSK-9) and low-density lipoprotein receptor (LDLR) gene expression. The expression of the PCSK9/LDLR/tumor necrosis factor-alpha (TNFα) gene in adipose tissue was measured in two groups of Zucker Diabetic Sprague Dawley (ZDSD) rats after Roux-en-Y gastric bypass (RYGB) surgery or 'SHAM' operation. There was lower PCSK9 (p = 0.02) and higher LDLR gene expression (p = 0.02) in adipose tissue in rats after RYGB. Weight change did not correlate with PCSK9 gene expression (r = -0.5, p = 0.08) or TNFα gene expression (r = -0.4, p = 0.1). TNFα gene expression was positively correlated with PCSK9 gene expression (r = 0.7, p = 0.001) but not correlated with LDLR expression (r = -0.3, p = 0.3). Circulating triglyceride levels were lower in RYGB compared to the SHAM group (1.1 (0.8-1.4) vs. 1.5 (1.0-4.2), p = 0.038) mmol/L with no difference in cholesterol levels. LDLR gene expression was increased post-bariatric surgery with the potential to reduce the number of circulating LDL particles. PCSK9 gene expression and TNFα gene expression were positively correlated after RYGB in ZDSD rats, suggesting that the modulation of pro-inflammatory pathways in adipose tissue after RYGB may partly relate to PCSK9 and LDLR gene expression.

Quantitative global lipidomics analysis of patients with ovarian cancer versus benign adnexal mass

Altered lipid metabolism has emerged as an important feature of ovarian cancer (OC), yet the translational potential of lipid metabolites to aid in diagnosis and triage remains unproven. We conducted a multi-level interrogation of lipid metabolic phenotypes in patients with adnexal masses, integrating quantitative lipidomics profiling of plasma and ascites with publicly-available tumor transcriptome data. Using Sciex Lipidyzer, we assessed concentrations of > 500 plasma lipids in two patient cohorts—(i) a pilot set of 100 women with OC (50) or benign tumor (50), and (ii) an independent set of 118 women with malignant (60) or benign (58) adnexal mass. 249 lipid species and several lipid classes were significantly reduced in cases versus controls in both cohorts (FDR < 0.05). 23 metabolites—triacylglycerols, phosphatidylcholines, cholesterol esters—were validated at Bonferroni significance (P < 9.16 × 10–5). Certain lipids exhibited greater alterations in early- (diacylglycerols) or late-stage (lysophospholipids) cases, and multiple lipids in plasma and ascites were positively correlated. Lipoprotein receptor gene expression differed markedly in OC versus benign tumors. Importantly, several plasma lipid species, such as DAG(16:1/18:1), improved the accuracy of CA125 in differentiating early-stage OC cases from benign controls, and conferred a 15–20% increase in specificity at 90% sensitivity in multivariate models adjusted for age and BMI. This study provides novel insight into systemic and local lipid metabolic differences between OC and benign disease, further implicating altered lipid uptake in OC biology, and advancing plasma lipid metabolites as a complementary class of circulating biomarkers for OC diagnosis and triage.

Effects of Yerba Mate tea (Ilex paraguariensis) on vascular endothelial function and liver lipoprotein receptor gene expression in hyperlipidemic rats

Yerba Mate tea (Mate), an infusion made from the leaves of the tree Ilex paraguariensis, is a widely consumed beverage in South America. This study was performed to investigate the effect of Mate tea on vascular endothelial dysfunction and liver lipoprotein receptor gene expression in hyperlipidemic rats, with the aim of gaining insight into its known lipid-lowering protective mechanisms. Sixty male Sprague–Dawley rats were randomly divided into five groups: a normal control group (NC), a high-fat diet group (HC), and three Mate tea-treated groups. In the NC group, rats were fed with standard diet while in the other groups the rats were fed a high-fat diet for 8weeks. In the Mate tea-treated groups, the rats were fed a high-fat diet supplemented with low, moderate or high concentrations of aqueous Mate tea extract for the final 4weeks. Compared to the HC group, aqueous Mate tea extract significantly reduced endothelin (ET) and thromboxane B2 (TXB2) levels and increased nitric oxide (NO) and 6-keto prostaglandin F1α (6-keto-PGF1α) levels in the blood, reduced the pathological damage of vascular endothelial cells, decreased intercellular adhesion molecule-1 (ICAM-1) protein expression in the thoracic aorta, and upregulated mRNA expression of hepatic low density lipoprotein receptor (LDLR) and scavenger receptor B1 (SR-B1). These findings indicate that Mate tea administration might have a regulatory effect on blood fat and endothelial function in hyperlipidemia rats. The mechanism may involve protecting vascular endothelial cell function and upregulating the expression of LDLR and SR-B1 genes, thereby inhibiting the occurrence of atherosclerosis.

RETRACTED: Plasma PCSK9 Levels and Clinical Outcomes in the TNT (Treating to New Targets) Trial: A Nested Case-Control Study

The purpose of this study was to investigate whether high levels of circulating proprotein convertase subtilisin kexin type 9 (PCSK9) would increase cardiovascular risk in statin-treated patients. Statins activate low-density lipoprotein (LDL) receptor gene expression, thus lowering plasma LDL levels. But statins also activate the expression of PCSK9, a secreted inhibitor of the LDL receptor, thereby limiting their beneficial effects. We have measured the plasma PCSK9 levels of 1,613 patients with stable coronary heart disease enrolled in the Treating to New Targets study, a randomized trial that compared the efficacy of high- versus low-dose atorvastatin. After a run-in period with atorvastatin 10 mg daily, patients were randomized to either continue with 10 mg or be up-titrated to 80 mg of atorvastatin, and followed during 5 years for major cardiovascular events (MCVEs). Circulating PCSK9 levels measured at randomization were predictive of clinical outcomes in the group randomized to remain on atorvastatin 10 mg (p = 0.039), but not in the group that intensified atorvastatin treatment to 80 mg (p = 0.24). Further, PCSK9 levels measured 1 year post-randomization did not change upon increase of the statin dose. PCSK9 levels predict cardiovascular events in patients treated with low-dose atorvastatin. (A Study to Determine the Degree of Additional Reduction in CV Risk in Lowering LDL Below Minimum Target Levels [TNT]; NCT00327691).

Effects of Mulberry (Morus alba L.) Extracts on Lipid Homeostasis in Vitro and in Vivo

The objective of this study was to investigate the lipid-lowering effects of mulberry water extracts (MWEs). To evaluate the hypolipidemic effect of MWEs, hamsters were fed with either high fat/cholesterol diets (HFCD) or HFCD supplemented with 1 and 2% MWEs for 12 weeks. Plasma total cholesterol (TC) and triglyceride (TG) levels of hamsters fed HFCD with MWEs were significantly reduced by about 30-37% and 16-35%, respectively, as compared to those without MWEs. Similar results were also measured in hepatic TC and TG of hamsters fed HFCD with MWEs. Low-density lipoprotein receptor (LDLR) gene expression and the uptake ability of low-density lipoprotein (LDL) in HepG2 cells were also upregulated by additions of MWEs. MWEs also decreased the gene expressions of enzymes involved in the TG and TC biosyntheses. Results suggest that hypolipidemic effects of MWEs are via an enhancement of LDLR gene expression and the clearance ability of LDL and a decrease in the lipid biosynthesis. Therefore, MWEs could be used as a natural agent against hyperlipidemia.

Protein kinase C activation stabilizes LDL receptor mRNA via the JNK pathway in HepG2 cells

LDL is the most abundant cholesterol transport vehicle in plasma and a major prognostic indicator of atherosclerosis. Hepatic LDL receptors limit circulating LDL levels, since cholesterol internalized by the liver can be excreted. As such, mechanisms regulating LDL receptor expression in liver cells are appealing targets for cholesterol-lowering therapeutic strategies. Activation of HepG2 cells with phorbol esters enhances LDL receptor mRNA levels through transcriptional and posttranscriptional mechanisms. Here, we show that 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-induced stabilization of receptor mRNA requires the activity of protein kinase C and is accompanied by activation of the major mitogen activated protein kinase pathways. Inhibitor studies demonstrated that receptor mRNA stabilization is independent of the extracellular signal-regulated kinase or p38(MAPK), but requires activation of the c-Jun N-terminal kinase (JNK). An essential role for JNK in stabilizing receptor mRNA was further confirmed through small interfering RNA (siRNA) experiments and by activating JNK through two protein kinase C-independent mechanisms. Finally, prolonged JNK activation increased steady-state levels of receptor mRNA and protein, and significantly enhanced cellular LDL-binding activity. These data suggest that JNK may play an important role in posttranscriptional control of LDL receptor expression, thus constituting a novel mechanism to enhance plasma LDL clearance by liver cells.

Role for LDL in estradiol-synthesis capacity of bovine ovarian follicles

Abstract The steroidogenic capacity of several tissues has been shown to be regulated by lipoproteins. However, the ability of the various lipoprotein classes to affect steroid production in general and estradiol-synthesis in particular has not been established in bovine ovarian cells. In previous studies, we described alterations in the lipoprotein profiles in follicles of different sizes and corresponding changes in lipoprotein-receptor gene expression. In the present study, the effect of low-density lipoprotein (LDL)-enriched medium on aromatization competence of whole ovarian follicles was determined in vitro. Gene expression of aromatase increased and that of selective-uptake receptors decreased in the presence of LDL. These results suggest a role for LDL availability in folliculogenesis.

Lowering Effect of an Isoflavone-Rich Fermented Soybean Extract on the Serum Cholesterol Concentrations in Female Rats, with or without Ovariectomy, but Not in Male Rats

We examined the effect of administering an isoflavone-rich fermented soybean extract (FSBE) on the serum cholesterol concentrations in male rats and in intact and ovariectomized (OVX) female rats. Dietary FSBE decreased the serum cholesterol concentrations in intact female and OVX rats, but did not affect the concentrations in male rats. Dietary FSBE increased the hepatic total and esterified cholesterol contents in the intact female rats, but decreased them in the OVX rats. This hypocholesterolemic effect was not a simple estrogenic effect because it has appeared in some reports that estrogen administration decreased serum cholesterol both male and female rats. Dietary FSBE increased the hepatic low-density lipoprotein receptor (LDLR) gene expression in the intact female rats as has previously been reported from many studies, but did not affect that of the OVX rats. Further investigation is needed into the hypocholesterolemic mechanism of FSBE.

Ultrasound-targeted microbubble destruction improves the low density lipoprotein receptor gene expression in HepG 2 cells

Ultrasound-targeted microbubble destruction had been employed in gene delivery and promised great potential. Liver has unique features that make it attractive for gene therapy. However, it poses formidable obstacles to hepatocyte-specific gene delivery. This study was designed to test the efficiency of therapeutic gene transfer and expression mediated by ultrasound/microbubble strategy in HepG 2 cell line. Air-filled albumin microbubbles were prepared and mixed with plasmid DNA encoding low density lipoprotein receptor (LDLR) and green fluorescent protein. The mixture of the DNA and microbubbles was administer to cultured HepG 2 cells under variable ultrasound conditions. Transfection rate of the transferred gene and cell viability were assessed by FACS analysis, confocal laser scanning microscopy, Western blot analysis and Trypan blue staining. The result demonstrated that microbubbles with ultrasound irradiation can significantly elevate exogenous LDLR gene expression and the expressed LDLRs were functional and active to uptake their ligands. We conclude that ultrasound-targeted microbubble destruction has the potential to promote safe and efficient LDLR gene transfer into hepatocytes. With further refinement, it may represent an effective nonviral avenue of gene therapy for liver-involved genetic diseases.

Ginsenoside Rb2 Upregulates the Low Density Lipoprotein Receptor Gene Expression through the Activation of the Sterol Regulated Element Binding Protein Maturation in HepG2 Cells

Ginsenosides, a group of Panax ginseng saponins, exert the lowering effects of plasma cholesterol levels in animals. We had reported earlier that ginsenoside Rb2 upregulate low-density lipoprotein receptor (LDLR) expression via a mechanism that is dependent of the activation of sterol response element binding protein 2 (SREBP-2) expression. This study was conducted to determine the effects of ginsenoside Rb2 on the expression of the hepatic LDLR expression at cellular levels using HepG2 cells, and to evaluate whether the sterol response element binding protein 1 (SREBP-l) was involved in the regulation of LDLR expression. Incubation of HepG2 cells in serum-free medium supplemented with cholesterol <TEX>$(10{\mu}g/ml)$</TEX> for 8 hours decreased the mRNAs of LDLR mRNA by <TEX>$12\%$</TEX> and SREBP-l mRNA by <TEX>$35\%$</TEX>. Ginsenoside Rb2 antagonized the repressive effects of cholesterol and increased both LDLR and SREBP-l mRNA expression to 1.5- and 2-fold, respectively. Furthermore, Western blot and confocal microscopic analyses with SREBP-l polyclonal antibody revealed that ginsenoside Rb2 enhanced the maturation of the SREBP-1 from the inactive precursor form in ER membrane to the active transcription factor form in nucleus. These results suggest that ginsenoside Rb2 upregulates LDLR expression via a mechanism that is dependent of the activation of not only SREBP-2 expression, but also SREBP-1 expression and maturation, and also indicate that the pharmacological value of ginsenoside Rb2 may be distinguished from that of lovastatin which is reported that it upregulate LDLR through SREBP-2 only, not through SREBP-1.

The farnesoid X receptor induces very low density lipoprotein receptor gene expression

The farnesoid X receptor (FXR) is a nuclear receptor activated by bile acids (BAs). In response to ligand-binding, FXR regulates many genes involved in BA, lipid, and lipoprotein metabolism. To identify new FXR target genes, microarray technology was used to profile total RNA extracted from HepG2 cells treated with the natural FXR agonist chenodeoxycholic acid (CDCA). Interestingly, a significant increase of transcript level of the very low density lipoprotein receptor (VLDLR) was observed. Our data, resulting from selective FXR activation, FXR RNA silencing and FXR-deficient mice, clearly demonstrate that BAs up-regulate VLDLR transcript levels via a FXR-dependent mechanism in vitro in human and in vivo in mouse liver cells.

The farnesoid X receptor induces very low density lipoprotein receptor gene expression

The farnesoid X receptor (FXR) is a nuclear receptor activated by bile acids (BAs). In response to ligand-binding, FXR regulates many genes involved in BA, lipid, and lipoprotein metabolism. To identify new FXR target genes, microarray technology was used to profile total RNA extracted from HepG2 cells treated with the natural FXR agonist chenodeoxycholic acid (CDCA). Interestingly, a significant increase of transcript level of the very low density lipoprotein receptor (VLDLR) was observed. Our data, resulting from selective FXR activation, FXR RNA silencing and FXR-deficient mice, clearly demonstrate that BAs up-regulate VLDLR transcript levels via a FXR-dependent mechanism in vitro in human and in vivo in mouse liver cells.

The low-density lipoprotein receptor is regulated by estrogen and forms a functional complex with the estrogen-regulated protein ezrin in pituitary GH3 somatolactotropes.

Estrogen regulates the function, growth, and proliferation of lactotropes in the pituitary. We report here that low-density lipoprotein (LDL) receptor (LDLR) gene expression and LDL uptake are strongly up-regulated by estrogen in pituitary somatolactotropic GH(3) cells. The uptake of LDL was significantly inhibited by the F-actin-severing drug, swinholide A, indicating that LDL uptake is dependent on the integrity of the cortical actin cytoskeleton in GH(3) cells. We examined whether the estrogen-inducible cytoskeletal linker protein, ezrin, interacts with the LDLR. The LDLR coimmunoprecipitated with ezrin, and fluorescently labeled LDL bound to regions of the cell membrane that colocalized with the active, phosphorylated form of ezrin (phosphoezrin). Evidence for a functional interaction between ezrin and the LDLR was obtained by transient transfection experiments using ezrin-green fluorescent protein (GFP) expression constructs. We observed that transient transfection of GH(3) cells with an ezrin N terminus-GFP dominant-negative construct prevented the uptake of LDL particles, whereas expression of GFP alone or an ezrin C terminus-GFP construct had no effect on LDL uptake. Transfection with the ezrin N terminus dominant- negative construct had no effect on the endocytosis of transferrin. Thus, estrogen stimulates the expression of ezrin and the LDLR in GH(3) cells, which interact physically and functionally to facilitate the endocytosis of LDL. We propose that the up-regulation and interaction of ezrin and the LDLR serves to augment the delivery of cholesterol and other lipids in support of the hypertrophic and proliferative response of cells to estrogen.

Bile Acids Enhance Low Density Lipoprotein Receptor Gene Expression via a MAPK Cascade-mediated Stabilization of mRNA

Recent studies have indicated that bile acids regulate the expression of several genes involved in bile acid and lipid metabolism as ligands for the farnesoid X receptor (FXR). We report here that bile acids are directly able to govern cholesterol metabolism by a novel mechanism. We show that chenodeoxycholic acid (CDCA) enhances low density lipoprotein (LDL) receptor gene expression in human cultured cell lines (HeLa, Hep G2, and Caco-2). The proteolytic activation of sterol regulatory element-binding protein-2 (SREBP-2), a major regulator for LDL receptor gene expression, is not affected by CDCA. Both deoxycholic acid and lithocholic acid as well as CDCA, but not ursodeoxycholic acid, increase the mRNA level for the LDL receptor, even when Hep G2 cells are cultured with 25-hydroxycholesterol, a potent suppressor of gene expression for the LDL receptor. Although it seems possible that FXR might be involved in genetic regulation, both reporter assays with a reporter gene containing the LDL receptor promoter as well as Northern blot analysis reveal that FXR is not involved in the process. On the other hand, inhibition of mitogen-activated protein (MAP) kinase activities, which are found to be induced by CDCA, abolishes the CDCA-mediated up-regulation of LDL receptor gene expression. We further demonstrate that CDCA stabilizes LDL receptor mRNA and that the MAP kinase inhibitors accelerate its turnover. Taken together, these results indicate that bile acids increase LDL uptake and the intracellular cholesterol levels through the activation of MAP kinase cascades in conjunction with a down-regulation of bile acid biosynthesis by FXR. This work opens up a new avenue for developing pharmaceutical interventions that lower plasma LDL by stabilizing LDL receptor mRNA.

Dexamethasone stimulates very low density lipoprotein (VLDL) receptor gene expression in differentiating 3T3-L1 cells

To characterize endocrine mechanisms of very low density lipoprotein (VLDL) receptor regulation we studied mouse adipocytic 3T3-L1 cells. Lipid filled adipocyte-like cells are formed during a 5–7 day time course in the presence of insulin, dexamethasone and isobutylmethylxanthine (IBMX). The VLDL receptor protein, in the form of its ≈120 and ≈100 kDa type I and type II isoforms, as well as binding of 125I-β-VLDL, was induced several-fold during differentiation. Among the three different constituents added to the culture medium only dexamethasone (1 μM), but not insulin or IBMX, induced a time- and dose-dependent increase of VLDL receptor expression. Inclusion of RU-486 (10 μM) blocked the stimulatory effect of dexamethasone on VLDL receptor mRNA and protein levels. 3.6 kb of the 5′-untranslated region representing the VLDL receptor promoter were cloned and sequenced, and the transcriptional start site was determined by primer extension to be located 574 bases upstream from the initiating methionine. To investigate the functionality of the promoter, luciferase reporter gene constructs for the region −181 to −3726 bases were assembled and transfected into 3T3-L1 cells. An increased reporter gene activity was recorded when comparing preconfluent cells to fully differentiated cells. Between day 0 and day 2 (48 h after transfection) reporter gene activity was induced by dexamethasone, but not by insulin or IBMX. RU-486 inhibited this stimulatory effect for all constructs tested. No classical glucocorticoid receptor (GR) response element was found in the sequenced region of the VLDL receptor promoter. Thus, an indirect stimulatory effect mediated via GR on VLDL receptor gene transcription is the most likely mechanism of VLDL receptor gene activation in differentiating 3T3-L1 cells.

Requirement of Sp1 and Estrogen Receptor Interaction in 17 -Estradiol-Mediated Transcriptional Activation of the Low Density Lipoprotein Receptor Gene Expression

Estrogen is one of the most important physiological regulators of low density lipoprotein receptor (LDLR) expression. Despite many studies conducted in animals and humans showing increased expressions of LDLR messenger RNA by hormone treatment, the molecular basis of the effect of estrogen on LDLR transcription has not been clearly elucidated. By using HepG2 cells that transiently express functional estrogen receptorα (ERα) and LDLR promoter constructs, we show that the specific interaction of ERα with the transcription factor Sp1 bound to the LDLR promoter is responsible for the activation of LDLR transcription by estrogen. We demonstrate that 1) mutations to abrogate the binding of Sp1 to its recognition sequences present in repeat 1 and repeat 3 elements of the LDLR promoter completely abolish the ERα-mediated activation of the LDLR promoter activity; 2) mutations that abolish the selective DNA-binding activity or inactivate the C-terminal transcription activation function (AF2) of ERα had no effect on the ability of ERα to activate LDLR transcription; however, transcriptional activation was completely lost by deletion of the N-terminal transcription activation region (AF1); 3) a subregion of AF1 (amino acids 67–139) was further identified to be important for ERα to activate the LDLR promoter; and 4) ERα enhanced the formation of Sp1-repeat 3 DNA complexes. We also show that mutation at the sterol-responsive element-1 site diminishes the activity of ERα on LDLR transcription, thereby suggesting that the sterol-responsive element-1-binding protein may interact with the Sp1-ERα complex to trans-activate LDLR gene transcription. This study for the first time provides a molecular basis for an understanding of the regulation of LDLR transcription by estrogens.

Requirement of Sp1 and estrogen receptor alpha interaction in 17beta-estradiol-mediated transcriptional activation of the low density lipoprotein receptor gene expression.

Estrogen is one of the most important physiological regulators of low density lipoprotein receptor (LDLR) expression. Despite many studies conducted in animals and humans showing increased expressions of LDLR messenger RNA by hormone treatment, the molecular basis of the effect of estrogen on LDLR transcription has not been clearly elucidated. By using HepG2 cells that transiently express functional estrogen receptor alpha (ERalpha) and LDLR promoter constructs, we show that the specific interaction of ERalpha with the transcription factor Sp1 bound to the LDLR promoter is responsible for the activation of LDLR transcription by estrogen. We demonstrate that 1) mutations to abrogate the binding of Sp1 to its recognition sequences present in repeat 1 and repeat 3 elements of the LDLR promoter completely abolish the ERalpha-mediated activation of the LDLR promoter activity; 2) mutations that abolish the selective DNA-binding activity or inactivate the C-terminal transcription activation function (AF2) of ERalpha had no effect on the ability of ERalpha to activate LDLR transcription; however, transcriptional activation was completely lost by deletion of the N-terminal transcription activation region (AF1); 3) a subregion of AF1 (amino acids 67-139) was further identified to be important for ERalpha to activate the LDLR promoter; and 4) ERalpha enhanced the formation of Sp1-repeat 3 DNA complexes. We also show that mutation at the sterol-responsive element-1 site diminishes the activity of ERalpha on LDLR transcription, thereby suggesting that the sterol-responsive element-1-binding protein may interact with the Sp1-ERalpha complex to trans-activate LDLR gene transcription. This study for the first time provides a molecular basis for an understanding of the regulation of LDLR transcription by estrogens.

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.

Sterol-independent, sterol response element-dependent, regulation of low density lipoprotein receptor gene expression

Stimulation with phorbol 12-myristate 13-acetate (PMA) and the calcium ionophore ionomycin increased native low density lipoprotein (LDL) receptor gene expression in the human leukemic T cell line Jurkat when cells were cultured in the absence of sterols and also increased nuclear accumulation of sterol regulatory element binding protein (SREBP)-1. PMA and ionomycin likewise increased LDL receptor mRNA levels when cells were cultured in the presence of suppressive concentrations of sterols, when neither SREBP-1 nor SREBP-2 was detectable in the nucleus. These findings indicated that mitogen-induced up-regulation of the LDL receptor gene could be independent of sterol-regulated transcription factors. The involvement of sterol regulatory element (SRE)-1 was analyzed by transfection of LDL receptor promoter constructs. Promoter fragments of either the 5′ 1472 or 142 base pairs induced reporter gene expression after mitogenic stimulation when cells were cultured in the absence or presence of sterols. Mutation of the SRE-1 sequence in either construct abolished sterol-mediated regulation of transcription. However, mutation of the SRE-1 sequence in the 1472 base pair promoter fragment did not alter mitogenic induction of transcription, whereas mutation of SRE-1 in the 142 base pair promoter fragment completely prevented up-regulation of transcription.▪ Taken together, these results demonstrate that the LDL receptor promoter contains at least one 5′ SRE-independent as well as an SRE-dependent response element. Furthermore, the data suggest that the SRE-dependent response may not involve the action of either SREBP-1 or –2. Thus, mitogen-induced transcription of the LDL receptor promoter is regulated by diverse sterol-independent mechanisms.—Makar, R. S. J., P. E. Lipsky, and J. A. Cuthbert. Sterol-independent, sterol response element-dependent, regulation of low density lipoprotein receptor gene expression.