Fourniers gangrene as Amyands hernia complication.

INTRODUCTION: Fourniers gangrene is a rare but fast deteriorating and serious condition with high mortality. In most cases, it is characterized as necrotizing fasciitis of the perineum and external genitals. Amyands hernia is a rare condition where the appendix is contained in the sac of an inguinal hernia. Inflammatory alterations in the appendix account only for 0.1 % of the cases when Amyands hernia is verified. Fourniers gangrene as a complication of a late diagnosis of appendicitis located in the inguinal canal is described in the literature as rare case reports.   Case report: The case report of a 70-year-old patient with Fourniers gangrene resulting from gangrenous appendicitis of Amyands hernia.  Conclusion: Fourniers gangrene as a complication of Amyands hernia is a rare condition. Only sporadic case reports thereof can be found in the literature. Because of the rarity of this pathology and the lack of randomized controlled studies, it is difficult to determine the optimal treatment according to the principles of evidence-based medicine. An appropriate approach for this condition appears to be the combination of guidelines developed in Amyands therapy according to Losanoff and Basson, along with the recommended “gold standard” therapy for Fourniers gangrene. This means early and highly radical surgical debridement, adequate antibiotic therapy and intensive care.

Lipopolysaccharide results in a marked decrease in hepatocyte nuclear factor 4 alpha in rat liver.

The acute-phase response can result in decreased liver-specific functions and death as a result of liver failure. We show here that lipopolysaccharide (LPS), an endotoxin that induces the acute-phase response, results in a marked decrease in the major isoforms of the transcription factor, hepatocyte nuclear factor 4 alpha (HNF-4 alpha), in livers of rats. HNF-4 alpha is a nuclear receptor that is critical for the expression of several liver-specific genes. This decrease in HNF-4 alpha is primarily the result of a posttranscriptional mechanism, because mRNA levels are normal, and there are no major changes in the splicing patterns. This decrease was of functional significance, because expression of a gene that is highly dependent on HNF-4 alpha, HNF-1 alpha, was reduced. Interleukin-1 beta (IL-1 beta) is a cytokine whose levels are increased in vivo in response to LPS. IL-1 beta resulted in a decrease in HNF-4 alpha levels in HepG2 cells. This IL-1 beta-induced decrease was likely caused by degradation via the proteasome, because it was prevented by the addition of the proteasome inhibitor, MG132. We conclude that the decrease in HNF-4 alpha that occurs in vivo after the administration of LPS may be the result of IL-1 beta-induced degradation, and likely contributes to the liver insufficiency that occurs. IL-1 beta antagonists or proteasome inhibitors might increase HNF-4 alpha protein levels in the acute-phase response, which could result in increased liver function and survival.

Novel nuclear target for thrombin: activation of the Elk1 transcription factor leads to chemokine gene expression.

Thrombin is primarily known for its role in homeostasis and thrombosis. However, this enzyme also plays important roles in wound healing and pathologic situations such as inflammation and tumorigenesis. Among the molecules stimulated by thrombin in these latter processes are the stress response proteins, chemokines. Chemokines are also known for their roles in inflammatory responses and tumor development. These correlative observations strongly suggest that chemokines may be mediators of some of thrombin's functions in these processes. Elucidation of the molecular mechanisms of stimulation of chemokines by thrombin may help to unravel the ways in which their expression can be modulated. Up-regulation of the chemokine 9E3/cCAF by thrombin occurs via its proteolytically activated receptor with subsequent transactivation of the epidermal growth factor receptor tyrosine kinase. This study shows that stimulation by thrombin very rapidly activates this chemokine at the transcriptional level, that 2 Elk1 binding elements located between -534 and -483 bp of the promoter are major thrombin response elements, that activation occurs via the Elk1 transcription factor, and that the latter is directly activated by MEK1/ERK2. The common occurrence of Elk1 binding domains in the promoters of immediate early response genes suggests that it may be characteristically involved in gene activation by stress-inducing agents. (Blood. 2000;96:3696-3706)

Analysis of protein dimerization and ligand binding of orphan receptor HNF4alpha.

Hepatocyte nuclear factor 4alpha (HNF4alpha) (NR2A1), an orphan member of the nuclear receptor superfamily, binds DNA exclusively as a homodimer even though it is very similar in amino acid sequence to retinoid X receptor alpha (RXRalpha), which heterodimerizes readily with other receptors. Here, experimental analysis of residues involved in protein dimerization and studies on a reported ligand for HNF4alpha are combined with a structural model of the HNF4alpha ligand-binding domain (LBD) (residues 137 to 384). When K300 (in helix 9) and E327 (in helix 10) of HNF4alpha1 were converted to the analogous residues in RXRalpha (E390 and K417, respectively) the resulting construct did not heterodimerize with the wild-type HNF4alpha, although it was still able to form homodimers and bind DNA. Furthermore, the double mutant did not heterodimerize with RXR or RAR but was still able to dimerize in solution with an HNF4alpha construct truncated at amino acid residue 268. This suggests that the charge compatibility between helices 9 and 10 is necessary, but not sufficient, to determine dimerization partners, and that additional residues in the HNF4alpha LBD are also important in dimerization. The structural model of the HNF4alpha LBD and an amino acid sequence alignment of helices 9 and 10 in various HNF4 and other receptor genes indicates that a K(X)(26)E motif can be used to identify HNF4 genes from other organisms and that a (E/D(X)(26-29)K/R) motif can be used to predict heterodimerization of many, but not all, receptors with RXR. In vitro analysis of another HNF4alpha mutant construct indicates that helix 10 also plays a structural role in the conformational integrity of HNF4alpha. The structural model and experimental analysis indicate that fatty acyl CoA thioesters, the proposed HNF4alpha ligands, are not good candidates for a traditional ligand for HNF4alpha. Finally, these results provide insight into the mechanism of action of naturally occurring mutations in the human HNF4alpha gene found in patients with maturity onset diabetes of the young 1 (MODY1).

Modulation of transcriptional activation and coactivator interaction by a splicing variation in the F domain of nuclear receptor hepatocyte nuclear factor 4alpha1.

Transcription factors, such as nuclear receptors, often exist in various forms that are generated by highly conserved splicing events. Whereas the functional significance of these splicing variants is often not known, it is known that nuclear receptors activate transcription through interaction with coactivators. The parameters, other than ligands, that might modulate those interactions, however, are not well characterized, nor is the role of splicing variants. In this study, transient transfection, yeast two-hybrid, and GST pulldown assays are used to show not only that nuclear receptor hepatocyte nuclear factor 4 alpha1 (HNF4alpha1, NR2A1) interacts with GRIP1, and other coactivators, in the absence of ligand but also that the uncommonly large F domain in the C terminus of the receptor inhibits that interaction. In vitro, the F domain was found to obscure an AF-2-independent binding site for GRIP1 that did not map to nuclear receptor boxes II or III. The results also show that a natural splicing variant containing a 10-amino-acid insert in the middle of the F domain (HNF4alpha2) abrogates that inhibition in vivo and in vitro. A series of protease digestion assays indicates that there may be structural differences between HNF4alpha1 and HNF4alpha2 in the F domain as well as in the ligand binding domain (LBD). The data also suggest that there is a direct physical contact between the F domain and the LBD of HNF4alpha1 and -alpha2 and that that contact is different in the HNF4alpha1 and HNF4alpha2 isoforms. Finally, we propose a model in which the F domain of HNF4alpha1 acts as a negative regulatory region for transactivation and in which the alpha2 insert ameliorates the negative effect of the F domain. A conserved repressor sequence in the F domains of HNF4alpha1 and -alpha2 suggests that this model may be relevant to other nuclear receptors as well.

MODY1 mutation Q268X in hepatocyte nuclear factor 4alpha allows for dimerization in solution but causes abnormal subcellular localization.

Recent studies have shown that mutations in the hepatocyte nuclear factor (HNF)-4alpha gene give rise to maturity-onset diabetes of the young, type 1 (MODY1). HNF-4, an orphan member of the nuclear receptor superfamily, contains a DNA-binding domain (DBD) and a putative ligand-binding domain (LBD) that can act independently of each other. The first MODY1 mutation identified creates a stop codon at amino acid 268 in the LBD of HNF-4 (Q268X) that leaves the DBD intact, suggesting that the mutant protein may retain some of the properties of the wild-type protein. To determine the functional properties of this mutant, we constructed HNF4.Q268X and tested it in vitro and in vivo for DNA binding, protein dimerization, and transactivation activity. Results of an electrophoretic mobility shift assay showed that HNF4.Q268X neither binds DNA alone nor binds it as a dimer with wild-type HNF-4 (HNF4.wt). In contrast, a co-immunoprecipitation assay showed that HNF4.Q268X is capable of dimerizing in solution with HNF4.wt. Transient transfection assays, however, indicated that HNF4.Q268X does not affect transactivation by HNF4.wt in vivo, supporting the argument against a dominant negative effect. Additional results suggest that the lack of a dominant negative effect could be due to a striking differential subcellular localization of the HNF4.Q268X protein: HNF4.Q268X could be extracted from transfected cells only when treated with SDS. Taken together, our results suggest that the MODY1 phenotype is due to a loss of functional HNF-4 protein that is aggravated in tissues that express relatively low amounts of HNF-4, such as pancreas.

Proposed mechanism for the stabilization of nuclear receptor DNA binding via protein dimerization.

Hepatocyte nuclear factor 4 (HNF-4) defines a new subgroup of nuclear receptors that exist in solution and bind DNA exclusively as homodimers. We recently showed that the putative ligand binding domain (LBD) of HNF-4 is responsible for dimerization in solution and prevents heterodimerization with other receptors. In this report, the role of the LBD in DNA binding by HNF-4 is further investigated by using electrophoretic mobility shift analysis. A comparison of constructs containing either the DNA binding domain (DBD) alone or the DBD plus the LBD of HNF-4 showed that dimerization via the DBD was sufficient to provide nearly the full DNA binding affinity of the full-length HNF-4. In contrast, dimerization via the DBD was not sufficient to produce a stable protein-DNA complex, whereas dimerization via the LBD increased the half-life of the complex by at least 100-fold. Circular permutation analysis showed that full-length HNF-4 bent DNA by approximately 80 degrees while the DBD bent DNA by only 24 degrees. Nonetheless, analysis of other constructs indicated that the increase in stability afforded by the LBD could be explained only partially by an increased ability to bend DNA. Coimmunoprecipitation studies, on the other hand, showed that dimerization via the LBD produced a protein-protein complex that was much more stable than the corresponding protein-DNA complex. These results led us to propose a model in which dimerization via the LBD stabilizes the receptor on DNA by converting an energetically favorable two-step dissociation event into an energetically unfavorable single-step event. Implications of this one-step model for other nuclear receptors are discussed.

Serine/threonine phosphorylation of orphan receptor hepatocyte nuclear factor 4.

We showed previously that hepatocyte nuclear factor 4 (HNF-4) defines a new subclass, Group IV, of nuclear receptors. In order to determine whether members of this subclass are phosphorylated, HNF-4 was overexpressed to high levels in insect cells using a baculovirus expression system. The baculovirus-expressed HNF-4 (HNF4.BV) was characterized and compared to HNF-4 overexpressed in transiently transfected mammalian (COS-7) cells (HNF4.COS). The results indicate that both HNF4.BV and HNF4.COS are phosphorylated although HNF4.BV was hypophosphorylated relative to HNF4.COS. Phosphoamino acid analysis showed that HNF-4 is phosphorylated mainly on serine and to a lesser extent on threonine residues. Phosphopeptide mapping revealed 13 phosphopeptides for HNF4.COS, only 9 of which were present in the HNF4.BV sample. DNA-binding studies also showed that HNF4.BV binds DNA with a lower specificity and affinity, as measured by the equilibrium dissociation constant (Kd), than does HNF4.COS. Partial proteolytic digestion experiments also revealed that HNF4.BV and HNF4.COS adopt somewhat different three-dimensional conformations. Since glycosylation of HNF4.BV was ruled out by a number of methods and since HNF-4 expressed in bacteria exhibited an even lower DNA-binding affinity than HNF4.BV, we propose that serine/theronine phosphorylation may play a role in the DNA-binding activity of HNF-4 and, therefore, possibly of other Group IV receptors as well.

The DNA binding domain of hepatocyte nuclear factor 4 mediates cooperative, specific binding to DNA and heterodimerization with the retinoid X receptor alpha.

We recently showed that hepatocyte nuclear factor 4 (HNF-4) defines a unique subclass of nuclear receptors that exist in solution and bind DNA elements as homodimers (Jiang, G., Nepomuceno, L., Hopkins, K., and Sladek, F. M. (1995) Mol. Cell. Biol. 15, 5131-5143). In this study, we show that the dimerization domains of HNF-4 map to both the DNA binding and the ligand binding domain. Whereas the latter is critical for dimerization in solution, the DNA binding domain mediates cooperative, specific binding to direct repeats of AGGTCA separated by one or two nucleotides. Whereas amino acid residues 117-125 (the T-box/third helix region) are insufficient for cooperative homodimerization and high affinity DNA binding, residues 126-142 (encompassing the A-box region) are required. Finally, in contrast to the full-length receptor, the DNA binding domain of HNF-4 is capable of heterodimerizing with that of the retinoid X receptor alpha but not with that of other receptors. These results indicate that the HNF-4 DNA binding domain is distinct from that of other receptors and that the determinants that prevent HNF-4 from heterodimerizing with RXR lie outside the DNA binding domain, presumably in the ligand binding domain.

Effect of dietary protein restriction on liver transcription factors.

The transcription of several genes that are preferentially expressed in the liver, including the serum albumin, transthyretin and carbamyl phosphate synthetase-I genes, is specifically decreased in animals consuming inadequate amounts of dietary protein. The high level of transcription of these genes in the liver is directed in part by a number of liver-enriched transcription factors, including hepatocyte nuclear factors (HNF)-1, -3, and -4, and proteins of the CCAAT/enhancer-binding protein (C/EBP) family. In the present study, we investigated the possibility that the co-ordinate decrease in transcription of the nutritionally sensitive genes in protein-deprived rats results from altered activity of one or more of the liver-enriched transcription factors. For HNF-4, Western blots indicated no change in the level of nuclear HNF-4 protein in liver of protein-deprived animals, whereas we observed a 40% reduction in the DNA binding activity of HNF-4 as measured by electrophoretic mobility shift assay (EMSA). Furthermore, the binding affinity of HNF-4 for DNA was unaltered by dietary protein deprivation, while the number of HNF-4 molecules able to bind to DNA (Bmax) was reduced, as determined by Scatchard analysis. This indicates that in the protein-restricted rats a portion of the pool of HNF-4 protein is inactivated or otherwise prevented from binding to DNA. The overall DNA binding activity of C/EBP alpha and beta was increased in protein-restricted animals. This change occurred in the absence of a change in the amount of the full-length forms of these two proteins, quantified by Western blotting. Interestingly, dietary protein restriction specifically increased the level of a truncated form of C/EBP beta (liver-enriched transcriptional inhibitory protein, LIP), which is a protein dominant negative inhibitor of C/EBP function. Analysis of HNF-3 DNA-binding activity by EMSA revealed that HNF-3 alpha and beta DNA binding was increased and that HNF-3 gamma DNA-binding activity was unchanged in protein-restricted animals. We also detected two apparently novel shift complexes with the HNF-3 probe by EMSA, both of which were decreased in protein-restricted animals. HNF-1 DNA-binding activity was increased by dietary protein restriction. We also examined the effect of protein restriction on the DNA-binding activity of two ubiquitous transcription factors, NF1 and Sp1. The DNA binding activity of the major NF1 isoforms was unchanged whereas the binding activity of Sp1 was increased in the protein-restricted animals. In summary, restriction of dietary protein resulted in a number of specific changes in the DNA-binding activity of various transcription factors. Because transcriptional activation typically involves the synergistic action of more than one transcription factor, small changes in the amount/activity of several factors, could have a strong net effect on the transcription of many genes.

Exclusive homodimerization of the orphan receptor hepatocyte nuclear factor 4 defines a new subclass of nuclear receptors.

Hepatocyte nuclear factor 4 (HNF-4), a highly conserved member of the steroid hormone receptor superfamily critical for development and liver-specific gene expression, is very similar to another superfamily member, retinoid X receptor alpha (RXR alpha), in overall amino acid sequence and DNA binding specificity. Since RXR alpha is known to heterodimerize with many other nuclear receptors, the formation of heterodimers between HNF-4 and RXR alpha was examined. With the electrophoretic mobility shift assay, coimmunoprecipitation, and transient transfection assays, it is shown that, unlike other nuclear receptors, HNF-4 does not form heterodimers with RXR alpha either in the presence or in the absence of DNA. We also show that in vitro-translated HNF-4 does not form heterodimeric complexes on DNA with a number of other receptors, including RXR beta, RXR gamma, retinoic acid receptor alpha, or thyroid hormone receptor alpha. To investigate the hypothesis that the lack of heterodimerization between HNF-4 and RXR alpha is due to a strong homodimerization activity of HNF-4, glycerol gradient sedimentation and kinetic analysis were used to show that HNF-4 is in fact a stable homodimer in solution. Finally, immunohistochemistry is used to show that the HNF-4 protein is found exclusively in the nuclei in both HepG2 cells, which express endogenous HNF-4, and transfected COS cells, which overexpress HNF-4. These findings lead us to propose that HNF-4 defines a new subclass of nuclear receptors which reside primarily in the nucleus and which bind DNA and regulate transcription as homodimers.

The orphan receptors COUP-TF and HNF-4 serve as accessory factors required for induction of phosphoenolpyruvate carboxykinase gene transcription by glucocorticoids.

Glucocorticoids stimulate hepatic phosphoenolpyruvate carboxykinase (PEPCK; EC 4.1.1.32) gene expression, thereby increasing the rate of gluconeogenesis. The effect of glucocorticoids on PEPCK gene expression is mediated by a set of promoter elements collectively referred to as the glucocorticoid response unit. The response unit spans a 100-bp segment and includes two glucocorticoid receptor binding sites (GR1 and GR2) and two accessory factor binding sites (AF1 and AF2), all of which are required for a maximal glucocorticoid response. The AF1 element also serves as a retinoic acid response element and may be involved in developmental and tissue-specific expression of the gene. In this study we report that COUP-TF and HNF-4, two orphan members of the nuclear receptor superfamily, bind to the AF1 element and function as accessory factors for the glucocorticoid response of the PEPCK gene.

Orphan receptor HNF-4 and bZip protein C/EBP alpha bind to overlapping regions of the apolipoprotein B gene promoter and synergistically activate transcription.

As the sole protein component of low density lipoproteins, apolipoprotein B (apoB) plays an important role in cholesterol metabolism. Previously, we found that the proximal promoter region of apoB (-81 to -52 relative to the start site) played a critical role in hepatocyte-specific gene expression and that that region contained overlapping binding sites for nuclear factors AF-1 (-81 to -62) and C/EBP (-69 to -52) (Metzger, S., Leff, T., and Breslow, J. L. (1990) J. Biol. Chem. 265, 9978-9983). In this study, we show that HNF-4, a member of the steroid hormone receptor superfamily, binds the AF-1 site on the apoB promoter and through it activates transcription in transient transfection assays in both liver and non-liver cell lines, HepG2 and HeLa, respectively. Mutational analysis of the AF-1/HNF-4 binding site indicated a correlation of HNF-4 binding and transcriptional activity. In addition, transient co-transfection experiments with HNF-4 and C/EBP alpha expression vectors showed that the two factors can synergistically activate transcription to levels more than 3-fold above the sum of either factor alone. Finally, using gel retardation analysis we show that purified HNF-4 and C/EBP proteins can concurrently occupy their overlapping binding sites on the apoB promoter in vitro. However, since the same system showed a lack of cooperative binding, we argue that an alternative mechanism is responsible for the synergistic effect of HNF-4 and C/EBP alpha on apoB gene transcription.

The expression pattern of a Drosophila homolog to the mouse transcription factor HNF-4 suggests a determinative role in gut formation.

A Drosophila gene, HNF-4(D), was selected by cross-hybridization with a probe to rat HNF-4 (hepatocyte nuclear factor 4), a steroid hormone receptor super-family member that plays an important role in liver-specific gene expression. The Drosophila gene matched the mouse gene in 60 out of 66 amino acids in the zinc finger DNA binding domain and in 140 out of 206 amino acids in the domain that specifies dimerization and ligand binding. HNF-4(D) is expressed in developing Drosophila embryos in mid-gut, fat bodies and malpighian tubules, a striking similarity to its limited expression in the adult intestine, liver and kidney of the mouse. Furthermore, Drosophila mutant that has a chromosome deletion spanning the HNF-4(D) locus fails to develop tissues where HNF-4(D) is expressed during late embryogenesis. These findings together with the earlier realization that the rat hepatocyte nuclear factor 3 (HNF-3) and forkhead, a Drosophila gene required for anterior and posterior gut formation, had virtually the identical DNA binding domain, lead us to speculate that a group of genes that participate in gut formation of invertebrates has survived in evolution to perform similar functions in mammals.

Orphan receptor HNF-4 and liver-specific gene expression.

Hepatocyte nuclear factor 4 (HNF-4), found in liver, kidney, and intestine, is a potent transcriptional activator that controls the expression of a wide variety of genes, including those involved in fatty acid and cholesterol metabolism, glucose metabolism, urea biosynthesis, blood coagulation, hepatitis B infections, and liver differentiation. HNF-4 is also a member of the steroid hormone receptor superfamily and has been highly conserved throughout evolution, suggesting that it might respond to an as yet unidentified ligand. In this presentation, some of the current findings regarding the role of HNF-4 in liver-specific gene expression are reviewed.

Disruption of a binding site for hepatocyte nuclear factor 4 results in hemophilia B Leyden.

Hemophilia B Leyden is an X chromosome-linked bleeding disorder characterized by very low plasma levels of blood coagulation factor IX (fIX) during childhood. After puberty, plasma fIX levels gradually rise to a maximum of 60% of normal, probably under the influence of testosterone. Single point mutations in the fIX promoter region of hemophilia B Leyden patients have been reported at -20, -6, -5, +8 and +13. In addition, one promoter mutation (G----C at -26) has been detected that abolishes fIX expression throughout life (M. Ludwig, personal communication). We examined how one of the hemophilia B Leyden mutations (T----A at -20) and the G----C mutation at -26 interfere with fIX gene transcription. We report that the wild-type promoter of the human fIX gene contains a binding site (at nucleotides -34 to -10) for hepatocyte nuclear factor 4 (HNF-4), a member of the steroid hormone receptor superfamily of transcription factors. The binding of HNF-4 is disrupted by both the T----A mutation at -20 and the G----C mutation at -26. Whereas HNF-4 transactivates the wild-type promoter sequence in liver (HepG2) and non-liver (HeLa) cell types quite well, it transactivates the -20 mutated promoter to only a limited extent and the -26 mutated promoter not at all. These data suggest that HNF-4 is a major factor controlling fIX expression in the normal individual and that its inability to bind efficiently to the -20 T----A and the -26 G----C mutated promoter sequence results in hemophilia. Further, the severity of the hemophilia phenotype appears to be directly related to the degree of disruption of HNF-4 binding and transactivation.

Murine chromosomal location of four hepatocyte-enriched transcription factors: HNF-3 alpha, HNF-3 beta, HNF-3 gamma, and HNF-4.

The genes for rat hepatocyte nuclear factors 3 and 4 (HNF-3 alpha, HNF-3 beta, HNF-3 gamma, and HNF-4) have been mapped in mouse by analysis of restriction fragment length polymorphisms in interspecific backcross mice. These hepatocyte-enriched transcription factors are positive-acting transcription factors with binding sites in regulatory regions of many genes expressed in hepatocytes. Both HNF-3 alpha, beta, and gamma and HNF-4 are also expressed in intestine. They have recently been implicated as potential participants in endodermal development from early gut cells because of their close homology to Drosophila genes, which themselves are expressed in the developing gut. Despite having similar functional roles and highly conserved DNA binding domains, the three loci from the Hnf-3 family of genes mapped to three different mouse chromosomes, suggesting that the Hnf-3 family has become widely dispersed during evolution and implying the necessity for independent activation of each member of the HNF-3 family.

Antagonism between apolipoprotein AI regulatory protein 1, Ear3/COUP-TF, and hepatocyte nuclear factor 4 modulates apolipoprotein CIII gene expression in liver and intestinal cells.

Apolipoprotein CIII (apoCIII), a lipid-binding protein involved in the transport of triglycerides and cholesterol in the plasma, is synthesized primarily in the liver and the intestine. A cis-acting regulatory element, C3P, located at -90 to -66 upstream from the apoCIII gene transcriptional start site (+1), is necessary for maximal expression of the apoCIII gene in human hepatoma (HepG2) and intestinal carcinoma (Caco2) cells. This report shows that three members of the steroid receptor superfamily of transcription factors, hepatocyte nuclear factor 4 (HNF-4), apolipoprotein AI regulatory protein 1 (ARP-1), and Ear3/COUP-TF, act at the C3P site. HNF-4 activates apoCIII gene expression in HepG2 and Caco2 cells, while ARP-1 and Ear3/COUP-TF repress its expression in the same cells. HNF-4 activation is abolished by increasing amounts of ARP-1 or Ear3/COUP-TF, and repression by ARP-1 or Ear3/COUP-TF is alleviated by increasing amounts of HNF-4. HNF-4 and ARP-1 bind with similar affinities to the C3P site, suggesting that their opposing transcriptional effects may be mediated by direct competition for DNA binding. HNF-4 and ARP-1 mRNAs are present within the same cells in the liver and intestine, and protein extracts from hepatic tissue, HepG2, and Caco2 cells contain significantly more HNF-4 than ARP-1 or Ear3/COUP-TF binding activities. These findings suggest that the transcription of the apoCIII gene in vivo is dependent, at least in part, upon the intracellular balance of these positive and negative regulatory factors.