AMP-activated protein kinase regulates gene expression by direct phosphorylation of nuclear proteins.

One of the primary functions of AMP-activated protein kinase (AMPK) is to regulate the metabolic pathways in response to reduced cellular energy charge. Most of the known targets of the kinase are cytoplasmic enzymes involved in both catabolic and anabolic metabolism. In addition, activation of AMPK in many cells results in changes in the pattern of gene expression. Although some of these effects are undoubtedly secondary responses to modified cellular metabolism, it is possible that in addition to its well-characterized function in the cytoplasm, AMPK also directly phosphorylates and regulates proteins involved in gene transcription. There are now several examples of transcription factors, cofactors and components of the transcriptional core machinery that are directly phosphorylated and regulated by AMPK. Here I review these examples and discuss the significance of AMPK activity in the nucleus.

Regulation of transcription by AMP-activated protein kinase: phosphorylation of p300 blocks its interaction with nuclear receptors.

AMP-activated protein kinase (AMP-kinase) modulates many metabolic processes in response to fluctuations in cellular energy status. Although most of its known targets are metabolic enzymes, it has been proposed that AMP-kinase might also regulate gene expression. Here we demonstrate that the transcriptional coactivator p300 is a substrate of AMP-kinase. Phosphorylation of p300 at serine 89 by AMP-kinase dramatically reduced its interaction, in vitro and in vivo, with the nuclear receptors peroxisome proliferator-activated receptor gamma, thyroid receptor, retinoic acid receptor, and retinoid X receptor, but did not affect its interaction with the non-nuclear receptor transcription factors E1a, p53, or GATA4. These findings indicate that the AMP-kinase signaling pathway selectively modulates a subset of p300 activities and represent the first example of a transcriptional component regulated by AMP-kinase. Our results suggest a direct link between cellular energy metabolism and gene expression.

A novel potent antagonist of peroxisome proliferator-activated receptor gamma blocks adipocyte differentiation but does not revert the phenotype of terminally differentiated adipocytes.

The antidiabetic thiazolidinediones, which include troglitazone and rosiglitazone, are ligands for the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma). Their antihyperglycemic effects seem to be linked to the regulation of PPARgamma-responsive genes. Here, we report the characterization of a specific PPARgamma antagonist that blocks several of the biological activities of the PPARgamma agonist rosiglitazone. PD068235 inhibited rosiglitazone-dependent PPARgamma transcriptional activity with an IC(50) of 0.8 microM and rosiglitazone-stimulated in vitro coactivator association. The role of PPARgamma in the initiation of differentiation is well documented. In this study, we used PD068235 as a tool to evaluate the functional role of PPARgamma in the maintenance of the terminally differentiated state. Treatment of confluent, growth-arrested 3T3-L1 preadipocytes with PD068235 blocked adipocyte differentiation induced by the standard adipogenic hormonal mixture (insulin/dexamethasone/isobutylmethylxanthin) and fully antagonized rosiglitazone-induced adipogenesis. In contrast, long-term treatment of terminally differentiated 3T3-L1 adipocytes with PD068235 did not induce any obvious morphological changes and had no effect on basal lipolysis rates. In addition, in fully differentiated adipocytes PD068235 did not alter the basal expression of PPARgamma target genes aP2 and CAP, but it effectively blocked rosiglitazone-induced expression of both genes. These results suggest that in terminally differentiated adipocytes, the PPARgamma activity is minimal and may not be required for the maintenance of PPARgamma target gene expression.

Differential activation of peroxisome proliferator-activated receptor-gamma by troglitazone and rosiglitazone.

The antidiabetic thiazolidinediones, which include troglitazone and rosiglitazone, are ligands for the nuclear receptor peroxisome proliferator-activated receptor (PPAR)-gamma and exert their antihyperglycemic effects by regulation of PPAR-gamma-responsive genes. We report here that PPAR-gamma activation by troglitazone depends on the experimental setting. Troglitazone acts as a partial agonist for PPAR-gamma in transfected muscle (C2C12) and kidney (HEK 293T) cells, producing a submaximal transcriptional response (1.8- to 2.5-fold activation) compared with rosiglitazone (7.4- to 13-fold activation). Additionally, troglitazone antagonizes rosiglitazone-stimulated PPAR-gamma transcriptional activity. Limited protease digestion of PPAR-gamma suggests conformational differences in the receptor bound to troglitazone versus rosiglitazone. Consistent with this finding, an in vitro coactivator association assay demonstrated that troglitazone-bound PPAR-gamma recruited the transcriptional coactivators p300 and steroid receptor coactivator 1 less efficiently than rosiglitazone-bound receptor. In contrast to these observations, troglitazone behaves as a full agonist of PPAR-gamma in 3T3L1 adipocytes. Two-dimensional protein gel electrophoresis demonstrated that troglitazone and rosiglitazone regulated distinct but overlapping sets of genes in several cell types. Thus, troglitazone may behave as a partial agonist under certain physiological circumstances and as a full agonist in others. These differences could be caused by variations in the amount of specific cofactors, differences in PPAR response elements, or the presence of different isoforms of PPAR-gamma.

Mitogen-activated protein kinase regulates transcription of the ApoCIII gene. Involvement of the orphan nuclear receptor HNF4.

The transcriptional regulation of the apoCIII gene by hormonal and metabolic signals plays a significant role in determining plasma triglyceride levels. In the current work we demonstrate that the apoCIII gene is regulated by the mitogen-activated protein (MAP) kinase signaling pathway. In HepG2 cells, repression of MAP kinase activity by treatment with the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase inhibitor PD98059 caused a 5-8-fold increase in apoCIII transcriptional activity. Activation of MAP kinase by phorbol ester treatment caused a 3-5-fold reduction in apoCIII transcription. The region of the apoCIII promoter responsible for this regulation was mapped in transiently transfected HepG2 cells to a 6-base pair element located at -740. The major protein binding to this site was identified as the nuclear hormone receptor HNF4. An increase in HNF4 mRNA and protein levels was observed in HepG2 cells after treatment with PD98059, indicating that the MAP kinase pathway regulates the expression of the HNF4 gene. These findings demonstrate that the apoCIII gene can be regulated by signals acting through the MAP kinase pathway and that this regulation is mediated, at least in part, by changes in the amount of HNF4.

Troglitazone, an antidiabetic agent, inhibits cholesterol biosynthesis through a mechanism independent of peroxisome proliferator-activated receptor-gamma.

Troglitazone is an antidiabetic agent of the thiazolidinedione family. It is generally believed that thiazolidinediones exert their insulin-sensitizing activity through activation of peroxisome proliferator-activated receptor-gamma (PPAR-gamma), a member of the steroid nuclear receptor superfamily. In the present study, we examined the effect of troglitazone on cholesterol biosynthesis in cultured Chinese hamster ovary (CHO) cells. Troglitazone inhibited biosynthesis of cholesterol, but not that of total sterols, in a dose-dependent manner, with a half-maximal concentration (IC50) value of 8 micromol/l. At 20 micromol/l, troglitazone inhibited cholesterol biosynthesis by more than 80%, resulting in the accumulation of lanosterol and several other sterol products. This inhibitory effect observed in CHO cells was also reproduced in HepG2, L6, and 3T3-L1 cells, suggesting that there is a common pathway for this troglitazone action. One hour after removal of troglitazone from the culture medium, disappearance of the accumulated sterols was accompanied by restored cholesterol synthesis, indicating that those accumulated sterols are precursors of cholesterol. PPAR-gamma reporter assays showed that PPAR-gamma activation by troglitazone was completely blocked by actinomycin D and cycloheximide. In contrast, the inhibition of cholesterol synthesis by troglitazone remained unchanged in the presence of the above compounds, suggesting that this inhibition is mechanistically distinct from the transcriptional regulation by PPAR-gamma. Like troglitazone, two other thiazolidinediones, ciglitazone and englitazone, exhibited similar inhibitory effect on cholesterol synthesis; however, other known PPAR-gamma ligands such as BRL49653, pioglitazone, and 15-deoxy-delta(12,14)-prostaglandin J2 showed only weak or no inhibition. The dissociation of PPAR-gamma binding ability from the potency for inhibition of cholesterol synthesis further supports the conclusion that inhibition of cholesterol biosynthesis by troglitazone is unlikely to be mediated by PPAR-gamma.

c-Jun N-terminal kinase phosphorylates peroxisome proliferator-activated receptor-gamma1 and negatively regulates its transcriptional activity.

The peroxisome proliferator-activated receptor-gamma (PPARgamma) transcription factor plays a pivotal role in adipocyte differentiation and metabolic regulation. The transcriptional activity of PPARgamma is positively modulated by ligand binding and negatively regulated by phosphorylation mediated by the MEK/ERK signaling pathway. The phosphorylation of mouse PPARgamma1 at Ser82 by ERK causes a decrease in both basal and ligand-dependent transcriptional activity. In this report we examined the ability of other mitogen-activated protein kinase family members to phosphorylate PPARgamma1. We demonstrate that in vitro, PPARgamma1 is efficiently phosphorylated by JNK/SAPK (c-Jun N-terminal kinase or stress-activated protein kinase) but only weakly phosphorylated by p38. In transfected 293T cells, PPARgamma1 is phosphorylated at Ser82 in response to known JNK activators such as UV irradiation and anisomycin treatment. This phosphorylation is not blocked by either the specific MEK inhibitor PD98059 or the p38 inhibitor SB203580, indicating that it is independent of the MEK/ERK and p38 signaling pathways. Finally, in transient transfection reporter assays, activation of JNK by anisomycin or by overexpression of MKK4 (the upstream JNK kinase) decreased ligand-dependent PPARgamma1 transcriptional activity. These results suggest that the activation of the JNK/SAPK pathway by extracellular signals, perhaps by inflammatory cytokines such as tumor necrosis factor-alpha, would result in a reduction of PPARgamma transcriptional activity and reduce the effects of PPARgamma ligands.

A common Hpa I RFLP of apolipoprotein C-I increases gene transcription and exhibits an ethnically distinct pattern of linkage disequilibrium with the alleles of apolipoprotein E.

Apolipoprotein (apo) C-I is a constituent of triglyceride-rich lipoproteins (TGRL) that interferes with their hepatic clearance. Functional polymorphism in the apoC-I gene has not been established. We determined that an Hpa I site variably present at -317 relative to the apoC-I gene is produced by a 4-bp CGTT insertion. The apoC-I Hpa I alleles showed an ethnically distinct pattern of linkage disequilibrium with the alleles of the adjacent apoE gene. The frequency of apoC-I Hpa I-positive (H2) with apoE varepsilon2 was 0. 98, without significant ethnic difference. In contrast, the frequency of H2 with apoE epsilon4 was 0.85 in European-Americans but only 0.55 in African-Americans (P < 0.001). The frequency of H2 with apoE epsilon3 was 0.02 in European-Americans and 0.08 in African-Americans (P < 0.001). African-American apoE epsilon3/epsilon3 carriers of apoC-I H2 had 19% lower fasting triglyceride levels than H1 homozygotes (P = 0.03) along with 18% higher HDL-cholesterol levels (P = 0.02). ApoB levels were 21% lower (P = 0.002). H2-allelic reporter-gene constructions showed 50% higher expression in transient transfection studies. We localized the source of this difference in expression to the CGTT insertion itself. Deletion studies of the H1 allele showed a negative transcriptional effect of the polymorphic region. An H1 oligodeoxynucleotide showed specific binding of a hepatoma-cell nuclear protein not evident with an H2 oligodeoxynucleotide. The H2 sequence may decrease the binding of a negatively acting transcription factor, leading to overexpression of apoC-I. This may produce a functional effect on lipoprotein levels but confirmation is needed in other populations.

Three isoforms of a hepatocyte nuclear factor-4 transcription factor with tissue- and stage-specific expression in the adult mosquito.

We cloned three isoforms of hepatocyte nuclear factor-4 (HNF-4) from the mosquito Aedes aegypti, designated AaHNF-4a, AaHNF-4b, and AaHNF-4c. AaHNF-4a and AaHNF-4b are typical members of the HNF-4 subfamily of nuclear receptors with high amino acid conservation. They differ in N-terminal regions and exhibit distinct developmental profiles in the female mosquito fat body, a metabolic tissue functionally analogous to the vertebrate liver. The AaHNF-4b mRNA is predominant during the previtellogenic and vitellogenic phases, while the AaHNF-4a mRNA is predominant during the termination phase of vitellogenesis, coinciding with the onset of lipogenesis. The third isoform, AaHNF-4c, lacks part of the A/B and the entire C (DNA-binding) domains. The AaHNF-4c transcript found in the fat body during the termination of vitellogenesis may serve as a transcriptional inhibitor. Both AaHNF-4a and AaHNF-4b bind to the cognate DNA recognition site in electrophoretic mobility shift assay. Dimerization of AaHNF-4c with other mosquito HNF-4 isoforms or with mammalian HNF-4 prevents binding to the HNF-4 response element. In transfected human 293T cells, AaHNF-4c significantly reduced the transactivating effect of the human HNF-4alpha1 on the apolipoprotein CIII promoter. Electrophoretic mobility shift assay confirmed the presence of HNF-4 binding sites upstream of A. aegypti vg and vcp, two yolk protein genes expressed in the female mosquito fat body during vitellogenesis. Therefore, HNF-4, an important regulator of liver-specific genes, plays a critical role in the insect fat body.

A novel compound that elevates high density lipoprotein and activates the peroxisome proliferator activated receptor.

In the current studies we describe the effects of PD 72953 and related compounds on lipoprotein levels in chow-fed male rats. After 2 weeks, 10 mg/kg of PD 72953 daily was as effective as 100 mg/kg gemfibrozil for elevating HDL-cholesterol. At 100 mg/kg, PD 72953 further elevated HDL-cholesterol to 232% of control levels, and was associated with increased HDL size and plasma apoE (169% of control), despite no change in hepatic apoE mRNA. ApoA-I rose transiently (at 1 week), but by 2 weeks only apoE remained elevated. PD 72953 dose-dependently reduced plasma apoB, VLDL-cholesterol, LDL-cholesterol, and triglyceride. Hepatic apoC-III mRNA reduction parallelled triglyceride lowering. After 1 week, 30 and 100 mg/kg per day PD 72953 reduced plasma apo-CIII levels by 30 and 34%, and triglycerides by 60 and 83%, respectively. PD 72953 treatment had no effect on triglyceride production rates; however, 125I-labeled VLDL apoB disappearance was enhanced. We compared PD 72953 to a structurally similar diacid, PD 69405, that also reduced VLDL and LDL, but had no effect on HDL elevation. Compared to PD 72953, PD 69405 further accelerated 125I-labeled VLDL apoB disappearance, decreased triglyceride production, and elevated the ratio of post-heparin hepatic to lipoprotein lipase activity. Whole animal studies, transient transfection studies in HepG2 cells, and chimeric receptor studies in kidney 293 cells suggest that PD 72953 is a ligand for the peroxisomal proliferation activated receptor alpha (PPARalpha), and PPARgamma. Overall, PD 72953 may act through a peroxisomal proliferation activated receptor and result in plasma triglycerides and apoB-containing lipoprotein reduction, while also raising HDL cholesterol. Reduced apoC-III may allow triglyceride-rich remnants to more efficiently bind and present substrate to peripheral tissue lipoprotein lipase, and therefore allow enhanced shedding of remnant phospholipid surface for HDL production.

Characterization of a dominant negative mutant form of the HNF-4 orphan receptor.

The HNF-4 orphan receptor is a member of the nuclear receptor family of transcription factors and a major regulator of genes involved in carbohydrate and lipid metabolism. As an initial step in characterizing the role of HNF-4 in the regulation of metabolism, we have generated a dominant negative form of HNF-4 (DN-HNF-4) that contains a defective DNA-binding domain. In gel mobility shift assays, DN-HNF-4 did not bind an oligonucleotide probe representing an essential HNF-4 binding site, C3P contained in the human apo CIII promoter, but did prevent the binding of two recombinant isoforms, HNF-4alpha1 and HNF-4alpha2, as well as naturally-occurring HNF-4. DN-HNF-4 had no effect on the binding of PPARgamma-RXRalpha heterodimers to a PPAR response element. In transfected HepG2 cells, DN-HNF-4 dramatically reduced constitutive transcriptional activity of the human apo CIII promoter and abolished the positive transcriptional activity caused by plasmids expressing either isoform of HNF-4. These results indicate that DN-HNF-4 is a selective dominant negative mutant which forms defective heterodimers with wild-type HNF-4, thereby preventing DNA binding and subsequent transcriptional activation by HNF-4.

Common genetic variation in the promoter of the human apo CIII gene abolishes regulation by insulin and may contribute to hypertriglyceridemia.

Overexpression of plasma apolipoprotein CIII (apo CIII) causes hypertriglyceridemia in transgenic mice. A genetically variant form of the human apo CIII promoter, containing five single base pair changes, has been shown to be associated with severe hypertriglyceridemia in a patient population. In animals and in cultured cells the apo CIII gene is transcriptionally downregulated by insulin. In this study we demonstrate that, unlike the wild-type promoter, the variant promoter was defective in its response to insulin treatment, remaining constitutively active at all concentrations of insulin. The loss of insulin regulation was mapped to polymorphic sites at -482 and -455, which fall within a previously identified insulin response element. Loss of insulin regulation could result in overexpression of the apo CIII gene and contribute to the development of hypertriglyceridemia. The variant apo CIII promoter is common in the human population and may represent a major contributing factor to the development of hypertriglyceridemia.

Hypolipidemic activity of select fibrates correlates to changes in hepatic apolipoprotein C-III expression: a potential physiologic basis for their mode of action.

For the last 30 years fibrates have been widely prescribed to treat human dyslipidemia. However, the primary mechanism by which they lower plasma lipid levels is still unknown. Studies with transgenic mice have suggested that changes in apoC-III expression levels have a dramatic influence on plasma triglyceride levels. These results suggested that fibrates could reduce lipid levels by lowering apoC-III gene expression. In the current studies, we sought to determine whether the selected fibrates, bezafibrate, clofibrate, fenofibrate, and gemfibrozil, could reduce hepatic apoC-III mRNA and plasma apoC-III levels. Chow-fed rats were orally gavaged daily with a dosing vehicle alone or with 100 mg/kg of each of the fibrates for 1 week and in addition with gemfibrozil for 2 weeks. Bezafibrate and fenofibrate lowered plasma triglyceride by approximately half and dramatically reduced hepatic apoC-III mRNA and plasma apoC-III levels. In contrast, clofibrate did not reduce plasma triglyceride levels and only partially reduced apoC-III mRNA and plasma protein levels. Gemfibrozil strongly reduced plasma triglyceride levels and had an intermediate but significant effect on apoC-III mRNA and plasma apoC-III levels. Some of the fibrates, especially gemfibrozil also reduced plasma apoC-II levels, an effect that could contribute to the observed triglyceride-lowering effect. In addition, the ratio of plasma apoE to plasma apoC-II plus apoC-III was strongly and inversely correlated with plasma triglyceride levels. As plasma apoE levels were not reduced in gemfibrozil-treated animals, this could also have contributed to the triglyceride-lowering effect of this fibrate. Fibrate-mediated triglyceride lowering was not the result of a decreased apoB or VLDL production and, therefore, suggested an enhanced VLDL remnant catabolism. Our results suggest that the mechanism by which fibrates lower plasma triglycerides is by reducing the level of hepatic apoC-III expression.

Transcriptional regulation of the apoC-III gene by insulin in diabetic mice: correlation with changes in plasma triglyceride levels.

Insulin-dependent diabetes mellitus (IDDM) is associated with elevated plasma triglyceride levels that normalize after insulin administration. The observation that overexpression of the apoC-III gene in transgenic mice can cause hypertriglyceridemia and other evidence implicating apoC-III in the regulation of triglyceride levels prompted us to examine whether apoC-III might be involved in the hypertriglyceridemia associated with IDDM. To this end, the regulation of apoC-III gene expression was studied in the streptozotocin-treated mouse model of IDDM. In the insulin-deficient diabetic state, these mice have elevated glucose and triglyceride levels and a 1.4- to 1.5-fold increase in hepatic apoC-III mRNA levels, by Northern analysis as well as quantitative solution hybridization RNase protection assay. Insulin treatment normalized the glucose and triglyceride levels and diminished hepatic apoC-III mRNA levels by 59%. Analysis of transcription rates using the nuclear run-on technique demonstrated that the changes in hepatic apoC-III mRNA levels were the results of changes in the transcriptional activity of the gene. To determine the role of insulin in the regulation of apoC-III transcription, HepG2 cells were transfected with an apoC-III reporter construct, and treated with different insulin concentrations. The results demonstrated that insulin treatment induced a dose-dependent down-regulation of apoC-III transcriptional activity. These data suggest that the apoC-III transcriptional changes seen in animals are caused by differences in insulin concentrations. Assuming that apoC-III mRNA levels reflect the synthesis and secretion of the protein, these results present the possibility that overexpression of the apoC-III gene could contribute to the hypertriglyceridemia observed in IDDM.

Apo CIII gene transcription is regulated by a cytokine inducible NF-kappa B element.

Overproduction of Apo CIII causes elevated plasma triglyceride levels in transgenic animals and is associated with hypertriglyceridemia in humans. The regulation of apo CIII production is likely to play an important role in controlling plasma triglyceride levels. As an initial step in determining the role of transcriptional regulation in the production of apo CIII and in triglyceride metabolism, we have begun to characterize the activity of specific transcriptional regulatory elements in the CIII promoter. In the current study, we have identified and characterized an NF-kappa B regulatory element located 150 nucleotides upstream from the transcriptional start site of the apo CIII gene. Purified NF-kappa B, as well as an NF-kappa B protein in HepG2 cell nuclear extracts, bound specifically to this sequence element. The hepatic protein was induced by phorbol ester (PMA), and reacted with antibodies to the p50 and p65 subunits of NF-kappa B. The NF-kappa B element conferred PMA and IL1-beta inducible transcriptional activity to a heterologous promoter/reporter construct when transfected into HepG2 cells. Analysis of the full length CIII promoter demonstrated that the inducible activity of the NF-kappa B element was suppressed by sequences in the apo CIII enhancer element located approximately 500 nucleotides upstream of the NF-kappa B binding site. A deletion removing the enhancer restored the PMA inducible activity of the NF-kappa B binding site. These results indicate that apo CIII gene expression is regulated by NF-kappa B, and suggest that apo CIII production may be modulated by cellular signals, like inflammatory cytokines, that activate NF-kB.

c-Myc does not require max for transcriptional activity in PC-12 cells.

The c-Myc proto-oncogene is a basic helix-loop-helix leucine zipper (b/HLH/LZ) protein that participates in cellular growth and differentiation. The expression of c-Myc mRNA is rapidly induced by nerve growth factor (NGF) and epidermal growth factor (EGF) in PC-12 pheochromocytoma cells. In most cell types, c-Myc forms a sequence-specific DNA binding complex with the stable, constitutively expressed Max. This complex can function as a transcriptional regulator. We show here that the expression of Max mRNA or protein was not detected in PC-12 cells. Nevertheless, treatment of PC-12 cells with NGF and serum caused an increase in the expression of the c-Myc protein and the transcription of a reporter gene linked to the Myc/Max DNA binding site. Transcription from the same reporter gene is stimulated by over-expression of c-Myc. These results suggest that c-Myc protein functions as a transcriptional regulator in PC-12 cells despite the lack of Max protein. Therefore, Myc/Max complexes may not be an absolute requirement for Myc-dependent gene expression.

Overexpression of apolipoprotein CII causes hypertriglyceridemia in transgenic mice.

We have generated transgenic mice expressing the human apolipoprotein CII (apoCII) gene under the transcriptional control of the human cytochrome P-450 IA1 (CYPIA1) promoter. Human apoCII transgenic (HuCIITg) mice exhibited significant basal expression of the transgene (plasma apoCII level = 26.1 +/- 4 mg/dl) and showed further induction of transgene expression after treatment with beta-naphthoflavone. Unexpectedly, HuCIITg mice were hypertriglyceridemic and human apoCII levels correlated strongly to triglyceride levels (R = 0.89, P < 0.0001). Triglyceride levels (mg/dl +/- SEM) were elevated compared to controls in both the fed (804 +/- 113 vs 146 +/- 18, P < 0.001) and fasted (273 +/- 39 vs 61 +/- 4, P < 0.001) states. HuCIITg mice accumulated triglyceride-rich very low density lipoproteins (VLDL) with an increased apoC/apoE ratio. Tracer kinetic studies indicated delayed clearance of VLDL-triglyceride, and studies using Triton inhibition of VLDL clearance showed no increase in VLDL production. Plasma from these mice activated mouse lipoprotein lipase normally and radiolabeled VLDL were normally hydrolyzed. However, HuCIITg VLDL showed markedly decreased binding to heparin-Sepharose, suggesting that apoCII-rich, apoE-poor lipoprotein may be less accessible to cell surface lipases or receptors within their glycosaminoglycan matrices. HuCIITg mice are a promising model of hypertriglyceridemia that suggests a more complex role for apoCII in the metabolism of plasma triglycerides.

Regulation of cytochrome P450 in cultured human colonic cells.

The consequences of altered cytochrome P450-dependent monooxygenase activities in colonic tissue are unknown. As an initial step toward elucidating underlying mechanisms that regulate cytochrome P450 levels in colonic epithelium, we have characterized CYP1A1(3) induction in cultured human colonic cells (Caco-2). The induction of CYP1A1 by several inducers was measured by enzymatic activity and immunoreactivity. 3-Methylcholanthrene, beta-naphthoflavone, and benz[alpha]anthracene were strong inducers of CYP1A1, benzo[alpha]pyrene induced CYP1A1 activity only weakly, while benzo[e]pyrene and phenobarbital were essentially inactive as inducers. The response of Caco-2 cells to beta-naphthoflavone is similar to that previously observed in vivo in the rat colonic epithelium. In addition, we have demonstrated that this induction results from an increase in CYP1A1 transcriptional activity. These results suggest that the Caco-2 cell line exhibits certain characteristics of cytochrome P450 activity observed in colonic epithelium and may serve as a model for studying cytochrome P450-dependent activity in this tissue. The identification of a colonic cell line that responds to xenobiotic inducers will be useful for examining mechanisms of cytochrome P450 induction in the colon and elucidating the potential role of this system in colonic carcinogenesis.

Nuclear factors AF-1 and C/EBP bind to the human ApoB gene promoter and modulate its transcriptional activity in hepatic cells.

ApoB-100, the major protein of low density lipoprotein, is produced primarily in the liver. The apoB promoter region from -86 to -52 binds two hepatic nuclear proteins. The protein that binds to the sequence between -86 to -61, called AF-1, was characterized and also shown to interact with the apoCIII, apoA-I, and apoCII promoters. We now show that the second protein, which by DNase I footprinting binds to the sequence between -69 and -52, is the heat stable hepatic factor C/EBP. The binding of this protein is competed by a known C/EBP-binding sequence in the transthyretin gene enhancer (TRR oligo 3). In addition, the protein protects the same region in DNase I footprinting as purified C/EBP. Mutants that affect AF-1 or C/EBP binding to the apoB promoter were created. These mutants were analyzed by the gel mobility shift assay and transient transfections into HepG2 cells. We have demonstrated that the two factors bind to overlapping sites and that they modulate apoB transcriptional activity.

A regulatory element in the ApoCIII promoter that directs hepatic specific transcription binds to proteins in expressing and nonexpressing cell types.

To better understand the mechanisms that determine cell type-specific gene expression, we have examined the transcriptional activity of a 13-nucleotide long sequence element, designated C3P, located in the promoter of the apoCIII gene. We demonstrate that this element is required for high levels of apoCIII gene expression in hepatic cells and is sufficient to determine hepatic specific expression when introduced into a heterologous promoter. A protein was identified in hepatic cell nuclear extracts, designated AF-1, that binds to this sequence and is presumably responsible for its transcriptional activity in hepatic cells. Even though the C3P element is not active in HeLa cells, a protein with C3P binding specificity was identified in HeLa cell nuclear extracts. While the HeLa protein is similar to the hepatic AF-1 in its binding specificity and relative abundance, it has approximately twice the molecular weight of the hepatic protein, indicating that they are different proteins or different forms of the same protein. A variety of murine tissue types, including those that do not express the apoCIII gene, were found to contain C3P binding proteins. We conclude that the cell type-specific activity of the C3P element is not due to the absence of C3P binding proteins in nonexpressing cells but is the result of qualitative differences in C3P binding proteins in different cell types.