Density Lipoprotein Receptor Gene Expression Research Articles

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.

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.

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.

Phorbol ester-induced low density lipoprotein receptor gene expression in HepG2 cells involves protein kinase C-mediated p42/44 MAP kinase activation

The signaling pathway involved in low density lipoprotein (LDL) receptor gene expression induced by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) was investigated in the human hepatoma HepG2 cell line. Treatment of HepG2 cells with 100 nM TPA resulted in an approximately 20-fold increase in LDL receptor mRNA level, as determined by RT-PCR, which peaked at 2-4 h of treatment and subsequently declined. The protein kinase C (PKC) inhibitors calphostin C and staurosporine prevented TPA-mediated LDL receptor mRNA induction. In contrast, TPA did not affect squalene synthase mRNA expression. Immunoblotting of cell extracts with isozyme-specific PKC antibodies revealed that HepG2 cells expressed PKC alpha, which was mainly cytosolic, and PKC beta, PK epsilon, and PKC zeta, all of which were present in both the cytosolic and particulate fractions. Treatment of HepG2 cells with 100 nM TPA resulted in translocation of cytosolic PKC alpha to the particulate fraction, with a maximum at 30 min-2 h of treatment, but was without effect on the subcellular distribution of the other isozymes. TPA treatment also led to activation of the mitogen-activated protein kinase (MAPK) ERK cascade. The specific MAPK pathway inhibitor PD98059 blocked TPA-induced ERK activation. Furthermore, pretreatment of cells with PD98059 inhibited TPA-induced LDL receptor mRNA induction. Moreover, pretreatment of cells with calphostin C inhibited TPA-mediated ERK activation and LDL receptor mRNA induction in a dose-dependent fashion. Based on a close kinetic correlation between PKC alpha translocation and ERK activation, and the effects of specific inhibitors, these findings suggest that translocation/activation of PKC alpha, and subsequent activation of the Raf-1/MEK/ERK MAPK cascade, represent key events in the transcriptional induction of LDL receptor gene by TPA in HepG2 cells.

Effects of gemfibrozil administration on very low density lipoprotein receptor mRNA levels in rabbits

To elucidate the regulation of very low density lipoprotein (VLDL) receptor gene expression, we administered to rabbits for 14 days gemfibrozil, a fibric acid derivative and a lipid lowering drug that is also included among peroxisome proliferators. VLDL receptor mRNA levels were examined by Northern blot analysis. The VLDL receptor mRNA levels in retroperitoneal adipose tissue and in gastrocnemius muscle were increased 6.9-fold and 3.7-fold, respectively, with gemfibrozil treatment, but no marked changes were observed in the heart, the organ in which VLDL receptor is most highly expressed. In the liver, VLDL receptor mRNA was not detected either before or after gemfibrozil administration. Lipoprotein lipase (LPL) and long-chain acyl coenzyme A synthetase (ACS) mRNA levels were also increased in parallel in adipose tissue. The enhanced expression of VLDL receptor mRNA may contribute to the increase of triglyceride-rich lipoprotein catabolism in peripheral tissues such as adipose tissue and muscles.

Activation of LDL receptor gene expression in HepG2 cells by hepatocyte growth factor

The effect of recombinant human hepatocyte growth factor (HGF) on low density lipoprotein (LDL) receptor gene expression was studied in the human hepatoma cell line HepG2. HepG2 cells were incubated with serum-free media in the presence and absence of HGF for various times and 125I-labeled LDL specific binding at 4 degrees C, uptake at 37 degrees C, and the levels of LDL receptor mRNA were measured. Incubation with HGF produced time- and concentration-dependent increases in 125I-labeled LDL binding (2-fold), uptake (2.5-fold), and LDL receptor mRNA (6-fold). HGF increased the rate of LDL receptor gene transcription 4- to 5-fold relative to that of several "house-keeping" genes as measured by nuclear run-on transcription. The half-life of LDL receptor mRNA, measured with actinomycin D, was not increased in HGF-treated cells. The stimulation of LDL receptor expression occurred independently of changes in cellular cholesterol or DNA biosynthesis or total cell protein. HepG2 cells were transiently transfected with plasmids bearing either three copies of repeats 2 and 3 (pLDLR(23)3LUC) or one copy of the LDL receptor promoter from -556 to +53 (pLDLR600LUC) linked to firefly luciferase. Incubation of pLDLR(23)3LUC, or pLDLR600LUC-transfected cells with HGF for 4 or 24 h at 37 degrees C produced a concentration-dependent increase in luciferase activity. A maximal stimulation of 3 to 6-fold was achieved for each construct at an HGF concentration of 100 ng/ml. In contrast, HGF had little or no effect on reporter activity in HepG2 cells transfected with a luciferase reporter plasmid bearing the HMG-CoA reductase promoter extending from -325 to +22. Thus, when compared to the native LDL receptor promoter, multiple copies of repeats 2 and 3 of the LDL receptor promoter can fully support activation of the luciferase reporter gene by HGF, demonstrating that the effect of HGF is mediated through the SRE-1. The lack of HGF effects mediated through the HMG-CoA reductase sterol regulatory element suggests, however, that sterol depletion may not be responsible for the induction of the LDL receptor promoter by growth factors. The signalling pathways or effectors responsible for activation of the LDL receptor and HMG-CoA reductase genes thus differ in their response to HGF. These data suggest that the level of SREBP's reaching the nucleus may be determined by as yet unidentified second messengers as well as by sterols.

Tyrosine kinase inhibitors potentiate the induction of low density lipoprotein receptor gene expression by hepatocyte growth factor

The role of tyrosine kinase, protein kinase C, cyclic nucleotide- and Ca 2+-calmodulin-dependent protein kinase second messenger pathways in the induction of LDL receptor gene expression by hepatocyte growth factor (HGF) was studied in the human hepatoma cell line Hep-G2. Incubation with media containing HGF increased the level of LDL receptor mRNA by 6.5-fold. Co-incubation with HGF and either of two tyrosine kinase inhibitors genistein (2.0–20.0 μg/ml) and herbimycin A (0.5–500.0 ng/ml) increased the level of LDL receptor mRNA above that observed with HGF alone by 40–60%. Incubation with HGF in the presence of the calmodulin antagonist W7 (10–30 μM) also super-induced the level of LDL receptor mRNA by nearly 230%. The protein kinase C and A inhibitors chelerythrine (0.1–10.0 μM) and H8 (0.5–5.0 μM), respectively, had no significant effects on the induction of LDL receptor mRNA by HGF. Taken together, these data suggest that tyrosine kinase, protein kinases C and A, and Ca 2+-calmodulin dependent protein kinase activities are not essential for activation of LDL receptor gene expression in Hep-G2 cells by HGF.

Transcriptional Modulators Affect in Vivo Protein Binding to the Low Density Lipoprotein Receptor and 3-Hydroxy-3-methylglutaryl Coenzyme A Reductase Promoters

Treatment of HepG2 cells with known effectors of low density lipoprotein receptor (LDLR) gene expression altered the in vivo pattern of protein-DNA interactions in the promoter. The observed changes are consistent with proteins binding in vivo to the sterol regulatory element (SRE), to Sp1-like sites, as well as to other regions. Protein bound to the SRE in all conditions, but the nature of the dimethyl sulfate reactivity changed depending on the physiological state of the cell. Hypermethylation within the SRE of the low density lipoprotein receptor promoter was observed when cells were treated with cholesterol synthesis inhibitors, insulin, or phorbol 12-myristate 13-acetate, suggesting that the SRE regulates this promoter through sterol-independent as well as sterol-dependent mechanisms. No significant changes were observed in binding to the Sp1-like sites, suggesting that differential binding to these sites does not play a role in altered transcription levels. Analysis of the 3-hydroxy-3-methylglutaryl coenzyme A reductase promoter also revealed protections that varied in a cell type-specific manner. Binding to the 3-hydroxy-3-methylglutaryl coenzyme A reductase SRE and putative nuclear factor 1 sites could be observed but varied little in different physiological conditions.

Regulation of low density lipoprotein receptor gene expression in HepG2 and Caco2 cells by palmitate, oleate, and 25-hydroxycholesterol

Our in vivo studies in mice have shown that LDL-receptor gene expression is regulated differently in both liver and intestine by dietary cholesterol and dietary saturated fat. While dietary cholesterol serves to regulate at transcriptional levels, dietary fatty acids do not. To study the mechanism of regulation of LDL-receptor by saturated fat and cholesterol at the cellular level, where any secondary effects of long-term feeding in vivo are minimized we used the cultured hepatoma and colon carcinoma cells, HepG2 and Caco2. LDL-receptor activity was determined by 125I-labeled LDL binding and uptake, LDL-receptor protein by Western blotting, LDL-receptor mRNA by RNase protection assay, and relative rates of LDL-receptor mRNA transcription by nuclear 'run-off' assay. Incubation of cells in lipoprotein-deficient serum (LPDS) for 48 h progressively induced LDL-receptor activity and LDL-receptor protein by 5- to 6-fold in HepG2 cells and 2- to 3-fold in Caco2 cells. Absolute levels of LDL-receptor mRNA and relative rates of LDL-receptor mRNA transcription also increased in parallel to the LDL-receptor activity and protein levels in both cell lines. These data suggest that LPDS induced the LDL-receptor gene by transcriptional mechanism. The suppressive effect of 25-hydroxycholesterol on LDL-receptor regulation was studied by incubating HepG2 and Caco2 cells grown either in 10% FCS or 10% LPDS for 24 h and then for 0-24 h with various doses of 25-hydroxycholesterol. In HepG2 cells, LDL-receptor activity and protein mass progressively decreased to 50% of zero time controls over 24 h. LDL-receptor mRNA levels and relative rates of transcription decreased in parallel. In Caco2 cells, 25-hydrocholesterol lowered LDL-receptor activity, mRNA, and transcription by approximately 35%. To examine the effects of palmitate on LDL-receptor regulation, palmitate was complexed with albumin. Palmitate decreased LDL-receptor activity by 25% in HepG2 cells without altering LDL-receptor mass, mRNA levels, or rates of mRNA transcription. Similarly, in Caco2 cells, palmitate decreased LDL-receptor activity and protein mass 30% of controls, but did not change LDL-receptor mRNA levels and/or rates of transcription. The combination of palmitate (0.8 mM) and 25-hydroxycholesterol (2.5-5 micrograms/ml) suppressed LDL-receptor activity by 65% in HepG2 cells and by 52% in Caco2 cells. However, LDL-receptor mRNA decreased by approximately 50% in HepG2 cells and 30-40% in Caco2 cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Non-sterol regulation of low density lipoprotein receptor gene expression in T cells

Non-sterol regulation of low density lipoprotein (LDL) receptor gene expression was examined in a mitogen-responsive human T cell line. Stimulation of the leukemic T cell line Jurkat with the phorbol ester phorbol 12-myristate 13-acetate (PMA) and the calcium ionophore ionomycin rapidly and transiently increased LDL receptor mRNA levels. Inhibition of protein synthesis with cycloheximide (CHX) or puromycin resulted in superinduction of LDL receptor mRNA levels by mitogenic stimulation. The increase in LDL receptor mRNA levels resulted from increased gene transcription rather than stabilization of mRNA half-life. Thus, similar results were obtained when reporter gene expression was assessed in Jurkat cells transfected with LDL receptor promoter constructs and mRNA half-life was not significantly altered by the stimuli. Neither mitogenic induction nor superinduction of LDL receptor mRNA levels in Jurkat cells was prevented by sterol downregulation of LDL receptor gene expression. The protein synthesis inhibitors CHX and anisomycin, but not puromycin, also directly stimulated LDL receptor mRNA levels, suggesting that these compounds could provide a signal required for LDL receptor gene transcription. Taken together, these data indicate that various non-sterol stimuli, including activation of protein kinase C, increases in intracellular calcium, inhibition of protein synthesis, and signals generated by the protein synthesis inhibitors CHX and anisomycin, induce LDL receptor gene expression. Thus, transcription of the LDL receptor gene is not only regulated by ambient sterols but also by a variety of influences that govern the various primary response or immediate early genes. These stimuli may play an important role in normal regulation of LDL receptor gene expression.

Cytokine regulation of low density lipoprotein receptor gene transcription in HepG2 cells

Elevated plasma levels of cytokines have been demonstrated in inflammatory, malignant, and infectious diseases. These disease states are often associated with abnormal lipid metabolism and reductions in plasma cholesterol levels. To determine if inflammatory cytokines could influence hepatic lipid metabolism, we evaluated low density lipoprotein (LDL) receptor function and gene expression in cytokine stimulated HepG2 cells, a hepatoblastoma-derived cell line which shares many functional similarities with normal hepatocytes. Tumor necrosis factor-alpha (TNF) and interleukin-1 beta (IL-1) increased LDL binding to HepG2 cells in a dose-responsive manner. Other cytokines including macrophage-colony stimulating factor, granulocyte macrophage-colony stimulating factor, and gamma-interferon had no significant effects on LDL binding. Increased binding in response to TNF or IL-1 was paralleled by increased steady-state levels of LDL receptor mRNA. Evaluation of LDL receptor mRNA half-life revealed no significant change in mRNA stability between control and TNF- or IL-1-stimulated cells. A fusion gene construct consisting of 1563 base pairs of the 5'-flanking DNA of the human LDL receptor gene was coupled to a luciferase reporter gene, transfected into HepG2 cells, and promoter activity was assayed after TNF and IL-1 challenge to the cells. TNF and IL-1 increased promoter activity 200-400%, while treatment with LDL inhibited promoter activity by 70-85%. TNF or IL-1 co-incubation with LDL could not override transcriptional inhibition by LDL. Pretreatment with cycloheximide prevented induction of LDL receptor mRNA by TNF, but not by IL-1, suggesting stimulation of LDL receptor transcription by TNF requires protein synthesis. We propose that TNF and IL-1, acting via distinct signal transduction pathways, increase surface LDL receptors by increasing gene transcription. Our findings suggest that cytokine-induced hypocholesterolemia may be related to TNF and/or IL-1 stimulation of hepatic LDL receptor gene expression and function.