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J. Biol. Chem., Vol. 278, Issue 51, 51813-51824, December 19, 2003

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Physical and Functional Interaction of Acyl-CoA-binding Protein with Hepatocyte Nuclear Factor-4^*

Anca D. Petrescu, Harold R. Payne, Amy Boedecker, Hsu Chao, Rachel Hertz¶, Jacob Bar-Tana¶, Friedhelm Schroeder, and Ann B. Kier||

From the Departments of Physiology and Pharmacology and Pathobiology, Texas A & M University, Texas Veterinary Medical Center, College Station, Texas 77843-4467 and the ^¶Department of Human Nutrition and Metabolism, Hebrew University Medical School, Jerusalem 91120, Israel

Received for publication, April 14, 2003 , and in revised form, October 1, 2003.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Although acyl-CoA-binding protein (ACBP) has been detected in the nucleus, the physiological significance of this observation is unknown. As shown herein for the first time, ACBP in the nucleus physically and functionally interacted with hepatocyte nuclear factor-4 (HNF-4), a nuclear binding protein that regulates transcription of genes involved in both lipid and glucose metabolism. Five lines of evidence showed that ACBP bound HNF-4 in vitro and in the nucleus of intact cells. (i) ACBP interaction with HNF-4 elicited significant changes in secondary structure. (ii) ACBP and HNF-4 were coimmunoprecipitated by antibodies to each protein. (iii) Double immunolabeling and laser scanning confocal microscopy (LSCM) of rat hepatoma cells and transfected COS-7 cells significantly colocalized ACBP and HNF-4 within the nucleus and in the perinuclear region close to the nuclear membrane. (iv) LSCM fluorescence resonance energy transfer determined an intermolecular distance of 53 Å between ACBP and HNF-4 in rat hepatoma cell nuclei. (v) Immunogold electron microscopy detected ACBP within 43 Å of HNF-4. These interactions were specific since ACBP did not interact with Sp1 or glucocorticoid receptor in these assays. The functional significance of ACBP interaction with HNF-4 was evidenced by mammalian two-hybrid and transactivation assays. ACBP overexpression in COS-7 or rat hepatoma cells enhanced transactivation of an HNF-4-dependent luciferase reporter plasmid by 3.2- and 1.6-fold, respectively. In contrast, cotransfection with antisense ACBP expression vector inhibited transactivation. LSCM of the individual triple fluorescent-labeled (HNF-4, ACBP, and luciferase) rat hepatoma cells showed a high correlation (r², 0.936) between the level of luciferase and the level of ACBP expression. In summary, ACBP physically interacted with HNF-4 in vitro and in intact cells, although ACBP expression level directly correlated with HNF-4-mediated transactivation in individual cells.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Acyl-CoA-binding protein (ACBP)¹ is a ubiquitous intracellular lipid-binding protein whose physiological function remains to be determined (reviewed in Refs. 1-3). Until recently, ACBP was thought to be primarily cytosolic and unique among the soluble intracellular lipid proteins in exhibiting very high affinity (K_d values of 0.5-10 nM) and exclusive specificity for long chain fatty acyl-CoAs (LCFA-CoAs) (1, 4-7). Based on the fact that ACBP exclusively binds LCFA-CoAs, it may participate in several aspects of LCFA-CoA metabolism.

First, ACBP may be involved in directly presenting LCFACoAs as substrates for lipid metabolic enzymes. A variety of studies in vitro suggest that ACBP extracts LCFA-CoAs from membranes (6) to increase the soluble LCFA-CoA pool available for intracellular transport (reviewed in Ref. 2). The cytosolic ACBP·LCFA-CoA complexes then interact with and present LCFA-CoA to acyltransferase enzymes involved in phospholipid synthesis in the endoplasmic reticulum (8, 9), lysophosphatidic acid synthesis in mitochondria (10), cholesteryl ester synthesis in the endoplasmic reticulum (11), and oxidation in mitochondria (10).

Second, ACBP may control the level of unbound LCFA-CoA available for interaction with regulatory sites on metabolic enzymes (e.g. acetyl-CoA carboxylase) and intracellular signaling proteins (e.g. protein kinase C) in the cytosol (reviewed in Refs. 12 and 13).

Third, recent data demonstrating the presence of significant amounts of ACBP in the nuclei of transfected cells overexpressing ACBP suggest that ACBP may also be involved in direct or ligand (LCFA-CoA)-dependent regulation of nuclear proteins that activate transcription of genes involved in lipid and glucose metabolism (14, 15). Several members of the nuclear receptor superfamily including the hepatocyte nuclear receptor 4 (HNF-4) (16-18), thyroid hormone receptor (TR) (19), and peroxisome proliferator-activated receptor- and - (PPAR- and -) (20, 21) interact with LCFA-CoAs. The relative order of affinities of these nuclear receptors for LCFA-CoAs is HNF-4 (K_d of 1.5-4 nM) >> TR (K_d of 120 nM) >> PPAR (displaces Wy14643). On this basis, it appears that only HNF-4 binds LCFA-CoAs with affinities in the physiological range of LCFACoA levels in the nucleus, <10 nM (17, 18). HNF-4 is a nuclear receptor with major roles in hepatocyte differentiation during liver development and in regulating the transcription of numerous target genes involved in lipid transport and metabolism (22, 23). Recently, a conditional gene knockout of HNF-4 in mice confirmed that HNF-4 is an important in vivo regulator of genes involved in lipid homeostasis including apolipoproteins (apoAI, -II, and -IV, apoB, apoCII and -III, and apoE), microsomal triglyceride transfer protein, cholesterol 7-hydroxylase (CYP7A), liver-fatty acid-binding protein (LFABP), and PPAR- (24). A study in which 9000 genes were analyzed by cDNA microarray technology in human hepatoma cells that overexpressed HNF-4 demonstrated that 26 of the 45 HNF-4-sensitive genes were related to lipid metabolism (25).

Although ACBP and HNF-4 both bind LCFA-CoAs with nanomolar affinities and have been reported to modulate lipid metabolism, it is not known if (i) ACBP interacts directly with HNF-4 at the molecular level, (ii) ACBP interacts directly with HNF-4 at the cellular level, and (iii) ACBP expression influences HNF-4 transcriptional functions.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Materials—Recombinant mouse ACBP was produced and purified as described (6). HNF--LBD was obtained as described earlier (16, 17). Fetal bovine serum, bovine serum albumin, protein A-Sepharose 4CL, FITC-goat anti-rat IgG, and protease inhibitor mixture were purchased from Sigma. Lab-Tek coverglass slides were purchased from Fisher. Mouse anti-firefly luciferase monoclonal antibody was from Novus Biologicals (Littleton, CO). Rabbit polyclonal antibodies against glucocorticoid receptor (GR) and Sp1 protein were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Gold-labeled polyclonal antibodies against rabbit IgG (Aurion, Wageningen, The Netherlands) were purchased through Electron Microscopy Sciences (Fort Washington, PA). Texas Red goat anti-rabbit IgG, TOTO-3 (a DNA staining dimeric cyanine fluorophore), and SlowFade kit were from Molecular Probes (Eugene, OR). FluoroLink Cy5-labeled goat anti-mouse IgG was from Amersham Biosciences. The mammalian expression vector pCi-neo, Renilla luciferase expression plasmid pRL-CMV, dual luciferase reporter assay system, and CheckMate mammalian two-hybrid system were from Promega (Madison, WI). LipofectAMINE 2000, used for DNA transfections, was purchased from Invitrogen. All reagents and solvents used were of the highest grade available and were cell culture tested as necessary.

CD of ACBP and HNF-4-LBD—Far-UV circular dichroic spectra of HNF-4-LBD and ACBP in 2 mM Tri-HCl, pH 8, containing 0.5% glycerol and 0.05 mM dithiothreitol were measured separately and in mixture. The CD measurements were performed with a J-710 Spectropolarimeter (Jasco, Baltimore, MD) using a 1-mm cuvette. Spectra were recorded from 250 to 195 nm at 50 nm/min with a time constant of 1 s and a bandwidth of 2 nm. For each CD profile an average of 10 scans was obtained. Percentages of various secondary structures in HNF-4-LBD, ACBP, and (HNF-4-LBD + ACBP) were calculated from CD spectra by using the CDstr program (26, 27). The CD spectrum of (ACBP + HNF-4-LBD) was compared with a theoretical spectrum obtained by summing the spectra of the HNF-4-LBD and ACBP recorded separately for each protein in a concentration equal to that in the mixture. Because of the molecular weight difference between ACBP (10,000 Da) and HNF-4-LBD (amino acids 132-455; 36,172 Da), 4 µM ACBP and 1.11 µM HNF-4-LBD solutions were used, respectively, such that the molar concentrations of amino acids were equivalent. For mixtures of the two proteins, 2 µM ACBP and 0.56 µM HNF-4-LBD were used in order to (i) maintain equivalent concentrations of amino acids from the two proteins and (ii) maintain the same total concentration of amino acids as was used for CD of the individual proteins.

ACBP and HNF-4-LBD Antisera for Coimmunoprecipitation, Western Blotting, and Immunocytochemistry—Rabbit polyclonal anti-rat ACBP and anti-rat HNF-4-LBD antisera were obtained as described previously (6, 9). The specificity of appropriate dilutions of the purified anti-ACBP antibodies for Western blotting was determined as described earlier (28). Anti-ACBP antisera did not cross-react with HNF-4-LBD, HNF-4. For fluorescence resonance energy transfer (FRET) determined by laser scanning confocal microscopy, pure fractions of anti-ACBP and HNF-4 IgGs were prepared using an Econo-Pac protein kit from Bio-Rad as described earlier (29, 30). The pure IgGs were then Cy3-labeled (anti-HNF-4 IgG) or Cy5-labeled (anti-ACBP IgG) using a Fluorolink-antibody Cy3 and Cy5 labeling kit from Amersham Biosciences according to the manufacturer's instructions.

Coimmunoprecipitation—The ability of polyclonal anti-ACBP or polyclonal anti-HNF-4-LBD antiserum to coimmunoprecipitate both proteins from mouse liver homogenate and rat hepatoma cells was tested. Mouse liver was diced in RIPA buffer containing protease inhibitor mixture (1 ml of buffer per 200 mg of liver tissue) and subjected to 30 strokes within a Dounce homogenizer. RIPA buffer consisted of 50 mM Tris, pH 8, 150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS (31). Unbroken cells and debris were removed by centrifugation at 600 x g for 5 min. Homogenates of mouse liver and hepatoma cells were incubated with antibodies against rat ACBP, rat-HNF-4-LBD, mouse GR, and mouse Sp1 protein, respectively, at 4 °C overnight. Protein A-Sepharose 4CL was blocked for nonspecific binding with 2% bovine serum albumin in RIPA buffer, added to the overnight immunoprecipitated complexes, and incubated for 2 h at 4 °C. After extensive washings with 500 mM NaCl in RIPA buffer, the beads were solubilized in 2x sample buffer for further SDS-PAGE separation and Western blotting. The immunoprecipitates were tested for the presence of ACBP, HNF-4, Sp1 protein, and GR by Western blotting as follows.

Western Blotting—Protein concentration was determined by BCA Protein Assay (Pierce). SDS-PAGE and protein transfer on nitrocellulose membranes were performed as described (17, 32). Primary antibodies used in Western blotting were rabbit anti-rat ACBP and anti-rat-HNF-4-LBD polyclonal antibodies prepared as described above. Specific proteins were visualized either by a colorimetric method utilizing alkaline phosphatase-conjugated goat anti-rabbit IgG and 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium substrate (Sigma) or by a chemiluminescent method employing horseradish peroxidase-conjugated goat anti-rabbit IgG and luminol/hydrogen peroxide substrates (Amersham Biosciences).

Detection of ACBP·HNF-4 Complexes by Immunoelectron Microscopy in Mouse Liver—Four-month-old mice were anesthetized with tribromoethanol and perfused through the left heart ventricle with 3% formaldehyde, 0.05% glutaraldehyde in 0.1 M sodium phosphate buffer, pH 7.3, with 3.2% sucrose. After 5 min of perfusion at 24 °C, the liver was collected, minced, and immersed in the same fixative for 1 h at 4 °C and then washed with 0.1 M sodium phosphate buffer containing 3.5% sucrose. The tissue was treated with 0.25% tannic acid for 1 h at 4 °C and with 2% uranyl acetate in 70% ethanol cooled at -35 °C for 40 min, and then dehydrated in an ethanol series and embedded in Lowicryl K4M resin. Ultrathin tissue sections (50-60 nm) were placed on Formvar-coated nickel grids and immunogold stained with anti-ACBP antiserum raised in rat (diluted 1:50-1:80) alone or in mixture with either anti-HNF-4 or anti-glucocorticoid receptor, raised in rabbit (diluted 1:150). These sections were washed and incubated with a mixture of goat anti-rat conjugated to 6-nm gold particles and goat anti-rabbit conjugated to 15-nm gold particles. All sections were post-stained very briefly with aqueous uranyl acetate and Reynold's lead citrate and examined with a Zeiss 10c transmission electron microscope.

Cell Culture—COS-7 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA). Rat hepatoma cells T-7 were generously provided by Dr. Charles Baum (University of Chicago, Chicago). The cells were grown in high glucose/Dulbecco's modified Eagle's culture medium containing 10% fetal bovine serum. 4-Well LabTek chamber slides were used for the immunofluorescence confocal microscopy experiments, and 6-well plates and 10-cm cell culture dishes for all the other purposes (e.g. transfection experiments).

Preparation of Cells for Indirect Immunofluorescence Microscopy— COS-7 and rat hepatoma cells grown in 4-well LabTek chamber slides were washed with Hanks' solution, fixed with 70:30 acetone/ethanol (v/v) for 30 min at -20 °C, and blocked against nonspecific binding with 10% fetal bovine serum in Hanks' solution for 1 h at room temperature. The cells were then incubated with primary antibodies for 1 h at room temperature, followed by incubation with fluorescently labeled secondary antibodies for another 1 h at room temperature. After extensive washing of unbound antibodies with Hanks' solution, the cells were mounted by the use of Slow Fade kit. Rabbit anti-rat HNF-4 polyclonal, rat anti-mouse ACBP polyclonal, and mouse anti-firefly luciferase monoclonal antisera were used as primary antibodies. Texas Red goat anti-rabbit IgG, FITC goat anti-rat-IgG, and FluoroLink Cy5-labeled goat anti-mouse IgG were used as secondary antibodies. In some experiments the nuclear areas of cells were defined by staining the DNA with TOTO-3, a dimeric cyanine fluorophore. Specificity of immunostaining was determined by deleting the primary antibody and using either one or both secondary antibodies. Several dilutions (1:20-1:200) of each primary and secondary antibody were tested and optimized. This minimized nonspecific adsorption of fluorescent antibodies, ensured separation of the fluorescent signals, and optimized fluorophore concentration to preclude self-quenching (33).

LSCM and Image Analysis—LSCM was performed on an MRC-1024 fluorescence imaging system (Bio-Rad) consisting of an Axiovert 135 microscope (Zeiss, New York) equipped with three independent low noise photomultiplier tube channels. The excitation light ( = 488, 568, and 647 nm) from a 15-milliwatt krypton-argon laser was delivered to the sample through x63 Zeiss Plan-Fluor oil immersion objective, numerical aperture 1.45. Texas Red and Cy3 were excited at 568 nm, and their emission was detected through an HQ 585/40 bandpass filter. FITC was excited with 488 nm light, and its emission was selected through a 540/30 filter. TOTO-3 (DNA stain) and Cy5 were excited with 647 nm light and their emission selected through a 680/32 bandpass filter. The conventional colors of emitted light were red (for Texas Red and Cy3), green (for FITC), and blue (for Cy5 and TOTO-3). Images were acquired and analyzed using LaserSharp (from Bio-Rad) and MetaMorph Image Analysis (from Advanced Scientific Imaging, Meraux, LA) software. Colocalization analysis was performed on an average of 20 cells from four different wells, and the results obtained are shown for a representative set of cells. LaserSharp version 3.0 software was used for colocalization analysis, and two values (colocalization coefficients) were calculated based on Pearson's correlation coefficient, which is a well established means of describing the degree of overlap between patterns or images (34). The software calculated the colocalization (coloc) coefficients for two different fluorophores, designated red (R) and green (G) herein, according to the following equations: C_red = R_i,coloc/ R_i and C_green = G_i,coloc/ G_i. In these equations R_i,coloc represents the sum of intensities of all red pixels that also have a green component; R_i is the sum of all the red pixels in the image; G_i,coloc represents the sum of intensities of all green pixels that have a red component; G_i is the sum of intensities of all green pixels in the image. The coefficients generated by LaserSharp have values between 0 and 1; a value of 0 indicates that there is no colocalization, whereas a value of 1.0 means there is complete colocalization. Similarly, for three channel images (designated red, green, and blue, respectively) analyses were performed not only to compare red and green colocalization but also green with blue and red with blue. These three channel analyses yielded three different colocalization coefficients, one for each pair of color combinations. The software allowed for independent background selection and subtraction before calculation of the coefficients. Fluorograms were generated from the entire image, displaying the intensity and distribution of different colored pixels within the merged image as a scattergram.

FRET Microscopy—In order to estimate the intermolecular distance between ACBP and HNF-4 molecules that were detected colocalized in confocal microscopy images, FRET between Cy3-labeled anti-HNF-4 IgG and Cy5-labeled anti-ACBP IgG was determined as described earlier (29, 30). Rat hepatoma cells were processed as for immunofluorescence microscopy, except that only primary antibodies that had been Cy3- and Cy5-labeled were used. Qualitatively, FRET from Cy3 to Cy5 was demonstrated by detection of sensitized emission of Cy5 (through the 680/32 bandpass filter) upon excitation of Cy3 at 568 nm, after correction of some bleed through of Cy3 emission through the same filter as follows. Fluorescence emission through 680/32 filter was recorded before and after photobleaching Cy5 at 647 nm for 3 min (the duration of photobleaching was optimized such that a good decrease in Cy5 fluorescence was obtained without affecting the emission intensity of Cy3). The image recorded after photobleaching Cy5 was the bleed through of Cy3 emission through PMT3 (with 680/32 bandpass filter). Fluorescence intensity of emission through this 680/32 filter before photobleaching Cy5 (FRET acceptor), being the sum resulted from sensitized emission of Cy5 and the bleed through of Cy5, was always higher than the one taken after Cy5 photobleaching, clearly indicating FRET from Cy3 to Cy5. Quantitative measurements for FRET efficiency estimation were carried out as described (29). FRET efficiency was calculated according to E = (I_post - I_pre)/I_post, where E is FRET efficiency; I_post is the acceptor (Cy3) fluorescence intensity after Cy5 photobleach, and I_pre is the acceptor (Cy3) fluorescence intensity before Cy5 photobleach. An average value of E was calculated from measurements of Cy3 fluorescence emission increase after Cy5 photobleaching in 50 different cells. The calculated E was further used to estimate R, the intermolecular distance between Cy3 and Cy5 (distance ultimately dictated in this experiment by the proximity between ACBP and HNF-4), according to E = 1/(1 + (R/R₀)⁶), where R₀, the distance for which a 50% FRET efficiency occurs, is known to be 50 Å for the Cy3/Cy5 FRET pair.

DNA Constructs and Plasmids Used in Transfection Assays—COS-7 and rat hepatoma cells were transfected with the following expression and reporter plasmids. A set of sense and antisense mouse ACBP expression vectors was constructed by inserting the 276-bp ACBP cDNA into an XhoI cloning site of pCI-neo mammalian expression vector purchased from Promega (Madison, WI). For an efficient repression of ACBP expression, an antisense ACBP vector was constructed using a pUC plasmid that contained the mouse pgk-1 gene promoter, as described (35). The HNF-4 expression vector pLEN4S, with the 3-kb rat HNF-41 cDNA under a human metallothionein-II promoter and human growth hormone 3'-untranslated region, was a gift from Dr. F. Sladek (University of California, Riverside). The ApoBLuc reporter vector, containing 4 inserts of the 85-47 HNF-4-responsive elements in the apoB gene promoter in front of the firefly luciferase cDNA was obtained from the same laboratory. For monitoring the transfection efficiencies, pRL-CMV (Promega, Madison, WI), the expression vector for Renilla luciferase was cotransfected with the other expression vectors.

Functional Interaction of ACBP with HNF-4 in Intact Cells, Transactivation Assay—COS-7 and rat hepatoma cells grown in 6-well culture plates were transfected with 1 µg each of ApoBLuc (reporter plasmid) and pLEN4S (HNF-4 expression vector), pCI-sACBP (sense ACBP expression vector), or pgk-aACBP (antisense ACBP RNA expression vector) per well. Renilla luciferase plasmid pRL-CMV (0.05 µg per well) was always cotransfected as internal standard for estimating the transfection efficiency. Transfections were performed with Lipofectamine 2000 reagent from Invitrogen according to the manufacturer's instructions.

The firefly luciferase activity, normalized to Renilla luciferase (as internal control for transfection efficiency), was determined by using the dual-luciferase reporter assay system from Promega (Madison, WI). Luminescence was read and measured with a Microlite ML3000 Microtiter plate luminometer equipped with BioLinx assay management software (Dynatech Laboratories, Inc., Chantilly, VA).

Appropriate controls were included as follows. (i) Controls for luciferase activity in cells transfected with ApoBLuc alone or ApoBLuc in the presence of empty vectors pLEN4, pCI-neo, and pgk, separate and together, were run to ensure that luciferase activity was an accurate reporter for HNF-4-driven expression. (ii) In experiments assessing the effect of ACBP on HNF-4-mediated transactivation, luciferase activity levels in cells transfected with sense or antisense ACBP expression vectors (i.e. pCI-sACBP or pgk-aACBP) were compared with cells transfected with empty vectors pCI-neo and pgk as negative controls.

Functional Interaction of ACBP with HNF-4 in Intact Cells, Mammalian Two-hybrid Assay—The interaction of ACBP with HNF-4 in intact cells was also examined by a mammalian two-hybrid system CheckMate Mammalian Two-Hybrid System from Promega (Madison, WI). The rat recombinant HNF-4 cDNA was from Dr. J. Bar-Tana (Hebrew University Medical School, Jerusalem, Israel). The mouse recombinant ACBP cDNA was prepared as described (6). Full-length HNF-4 and ACBP cDNA fragments were amplified by PCR using primer sets containing specific restriction cloning sites as follows: (i) for ACBP, 29-mer 5'-CTGGATCCGTATGTCTCAGGCTGAATTTG-3' and 33-mer 5'-GGTCTAGATTATATTCCGTATTTCTTCTTTAGC-3'; (ii) for HNF-4, 30-mer 5'-CAGGATCCACATGGACATGGCTGACTACAG-3' and 28-mer 5'-GCTCTAGACTAGATGGCTTCCTGCTTGG-3'. PCR fragments were restriction-digested with BamHI/XbaI and ligated to pACT and pBIND vectors to produce pACT/HNF-4 and pBIND/ACBP, pACT/ACBP and pBIND/HNF-4. The four constructs were purified and sequenced at their 5' junctions to verify that the introduced cDNAs were in the correct reading frame. Two separate sets of experiments were performed to ensure there was no vector preference for either cDNA. COS-7 cells, were cotransfected with pACT/HNF-4, pBIND/ACBP in a first set of experiments, and with pACT/ACBP, pBIND/HNF-4 in a second set, together with the firefly luciferase reporter plasmid pG5luc, using LipofectAMINE 2000 (from Invitrogen). Renilla luciferase plasmid, pRL-CMV (Promega, Madison, WI), was used as internal control to monitor transfection efficiency at all times. Transfected COS-7 cells were cultured for 24 h, harvested, and lysed. Renilla and firefly luciferase activities were measured by using Dual Reporter Assay System (Promega, Madison, WI) and an ML3000 Microtiter Plate luminometer equipped with BioLinx assay management software (Dynatech Laboratories Inc., Chantilly, VA).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
ACBP·HNF-4 Complex Formation in Vitro, Circular Dichroism—To determine whether ACBP and HNF-4-LBD physically interact to alter conformation, CD was used to determine the secondary structure of these proteins obtained individually and in combination. The shape of the far-UV CD spectrum of ACBP indicated the presence of high amounts of -helix (Fig. 1, solid triangles). Quantitative analysis of multiple CD spectra of ACBP showed that ACBP contains nearly half of its polypeptide chain as -helix (48%) structure, with very little -sheet (2%) in its secondary structure. In contrast, CD spectra of HNF-4-LBD in buffered aqueous solution indicated that -helix structures represented only a minor component of this protein (Fig. 1, solid circles). This was confirmed by quantitative analysis of multiple CD spectra that showed HNF-4-LBD as predominantly -strand structure (27.3%) with low content of -helices (3.3%) (Table I).

View larger version (19K): [in this window] [in a new window]   FIG. 1. Circular dichroic spectrum of ACBP in the presence of HNF-4-LBD indicates complex formation. Far-UV CD spectra of ACBP (black triangles), HNF-4-LBD (black circles), and a mixture of ACBP and HNF-4-LBD (white circles).

View this table: [in this window] [in a new window]   TABLE I Secondary structure of ACBP, HNF4-LBD, and complexes of these two proteins

Detection of ACBP/HNF-4 Interaction in Rat Hepatoma Cells and Mouse Liver, Coimmunoprecipitation—To investigate further the possibility that ACBP binds HNF-4-LBD to form an ACBP·HNF-4 complex, the homogenate of mouse liver tissue was treated with polyclonal antisera to ACBP or HNF-4-LBD in a coimmunoprecipitation assay as described under "Experimental Procedures." Anti-ACBP serum immunoprecipitated a high amount of ACBP from mouse liver homogenate, as expected (Fig. 2, lane 1, row a) and also a lower but significant amount of HNF-4 (Fig. 2, lane 1, row b). The coimmunoprecipitation of ACBP and HNF-4 was also demonstrated with anti-HNF-4 serum which produced high amounts of HNF-4 (Fig. 2, lane 2, row b) but also detectable amounts of ACBP (Fig. 2, lane 2, row a) from the liver homogenate. As controls for the specificity of ACBP/HNF-4 coimmunoprecipitation, polyclonal antibodies against two other nuclear proteins with roles in transcription regulation, i.e. Sp1 protein and glucocorticoid receptor (a nuclear receptor with modular structure, similar to HNF-4), were used to test coimmunoprecipitation with ACBP (Fig. 2). Sp1 protein was not detected in immunoprecipitates generated with anti-ACBP (Fig. 2, lane 1, row c), even though it was found present in liver homogenate (Fig. 2, lane 5, row c). Immunoprecipitates obtained with anti-Sp1 (Fig. 2, lane 3) contained Sp1 (Fig. 2, lane 3, row c) but not ACBP or HNF-4 (Fig. 2, lane 3, rows a and b, respectively). GR was not detected after anti-ACBP immunoprecipitation (Fig. 2, lane 1, row d). Although a trace amount of ACBP was detected after anti-GR immunoprecipitation (Fig. 2 lane 4, row a), the fact that anti-ACBP did not immunoprecipitate GR suggested that this was nonspecific. These results were consistent with ACBP directly and specifically interacted with HNF-4 in mouse liver as well as in hepatoma cells in culture (data not shown for hepatoma cells).

View larger version (22K): [in this window] [in a new window]   FIG. 2. Coimmunoprecipitation of ACBP and HNF-4-LBD from liver homogenates. Immunoprecipitates (IP) were obtained with antibodies as specified: anti-ACBP, lane 1; anti-HNF-4, lane 2; anti-Sp1 protein, lane 3; and anti-GR, lane 4. Lane 5 shows the input, i.e. liver homogenate that has been used for immunoprecipitation. Western blots (WB) are shown for ACBP (row a), HNF-4 (row b), Sp1 (row c), and GR (row d) (the latter two were used as control for the specificity of HNf-4/ACBP coimmunoprecipitation).

View larger version (71K): [in this window] [in a new window]   FIG. 3. Intracellular colocalization of ACBP with HNF-4 and GR in mouse hepatocyte nuclei by immunoelectron microscopy. A and B, antigenic sites of ACBP were labeled with 6-nm gold particles as described under "Experimental Procedures." Antigenic sites of HNF-4 and GR were labeled with 15-nm gold particles in A and B, respectively. The bar is 0.5 µm, and the insets are x4 magnifications of the details indicated by arrows.

View larger version (50K): [in this window] [in a new window]   FIG. 4. Confocal microscopy colocalization of ACBP and HNF-4 in rat hepatoma cells. A, fluorescence image of HNF-4 labeled indirectly with Texas Red; B, green fluorescence image of ACBP labeled indirectly with FITC; C, the overlay of HNF-4 and ACBP fluorescence images in A and B; D, selected pixels with high fluorescence intensity for both red and green labels are shown in yellow.

View larger version (74K): [in this window] [in a new window]   FIG. 5. Confocal FRET microscopy demonstrates ACBP binding HNF-4 in rat hepatoma cell nuclei. Rat hepatoma cells were labeled with Cy3 anti-HNF-4 and Cy5 anti-ACBP. A, Cy3 (FRET donor) was excited at 568 nm and its emission detected through a HQ598/40 bandpass filter. B, Cy5 (FRET acceptor) was excited at 647 nm and its emission detected through a 680/32 bandpass filter. C, sensitized emission of Cy5 plus bleed through of Cy3 was measured through 680/30 bandpass filter upon excitation at 568 nm. D, same as in A but after photobleaching of Cy5 at 647 nm. E, no emission of Cy5 at 680/32 nm was detected after photobleaching with excitation at 647 nm for 3 min. F, a low level of fluorescence emission at 680/32 nm with excitation of Cy3 at 568 nm indicated some bleed through of Cy3 fluorescence through the 680/32 bandpass filter. G, same as in A but with image processing and color representation of emission fluorescence intensity from zero intensity (blue) to medium (yellow) and high (red) level. H, sensitive emission of Cy5 through the 680/32 filter with excitation of Cy3 at 568 nm, obtained by subtracting fluorescence intensity shown in F from that shown in C (correction for Cy3 bleed through 680/32 filter). I, same as in D but with colored image processing representing zero emission intensity in blue, medium intensity in yellow, and high intensity levels in red. Increase in donor fluorescence intensity (I, red pixels pointed out by arrows) after acceptor photobleaching allowed calculation of FRET efficiency and determination of the ACBP and HNF-4 intermolecular distance (53 Å).

View larger version (12K): [in this window] [in a new window]   FIG. 6. Mammalian two-hybrid assay of ACBP interaction with HNF-4 In this assay the level of firefly luciferase expression is directly proportional with the affinity between ACBP and HNF-4. Firefly luciferase activity has been normalized to Renilla luciferase activity and expressed in relative light units. Group 1, COS-7 cells were transfected with pACT/ACBP and pBIND/HNF-4 (Exp. A) or with pACT/HNF-4 and pBIND/ACBP (Exp. B); group 2, negative control, COS-7 cells were transfected with pACT and pBIND/HNF-4 (Exp. A) or pBIND/ACBP (Exp. B); group 3, negative control, COS-7 cells were transfected with pBIND and pACT/ACBP (Exp. A) or pACT/HNF-4 (Exp. B); group 4, positive control, COS-7 cells were transfected with pACT/myoD and pBIND/ID (Exp. A and B).

View larger version (14K): [in this window] [in a new window]   FIG. 7. ACBP and HNF-4 expression in transfected cells estimated from Western blots. A, ACBP and HNF-4 bands detected in COS-7 cells, nontransfected (lane 1) or transfected with pLEN4S (HNF-4 expression vector), pCI-sACBP (ACBP expression vector), and pgk-aACBP (ACBP antisense RNA expression plasmid) (lanes 2-5). B, ACBP and HNF-4 bands detected in rat hepatoma cells nontransfected (lane 1) or transfected with pCI-sACBP, pgk-aACBP (lanes 2 and 3).

These Western blotting data were confirmed by indirect immunofluorescence microscopy. COS-7 cells either nontransfected (Fig. 8A) or transfected with HNF-4 expression vector, pLEN4s (Fig. 8B), sense ACBP expression vector, pCI-sACBP (Fig. 8, D-F), and antisense ACBP vector, pgk-aACBP (Fig. 8C), were fixed and labeled with Texas Red (red fluorescence) for HNF-4 and with FITC (green fluorescence) for ACBP. The ACBP level was detectable in nontransfected COS-7 cells (Fig. 8A), increased in numerous cells (80%) upon transfection with pCI-sACBP alone (Fig. 8D) or together with HNF-4 expression vector (Fig. 8, E and F), and decreased in most of the cells (90%) cotransfected with antisense ACBP vector, pgk-aACBP (Fig. 8C). HNF-4 expression in COS-7 cells assessed by confocal microscopy demonstrated that nontransfected cells did not exhibit any HNF-4 (Fig. 8A), although pLEN4S (HNF-4) vector-transfected COS-7 cells expressed HNF-4 in 20% of cells (Fig. 8, B, C, E, and F) in contrast to the ectopic expression of ACBP, in 80% of the cells. This difference in expression efficiency was apparently determined not only by the transfection efficiency but also by the promoter strength. When cotransfected with HNF-4 and sense ACBP expression vectors, some cells expressed both proteins (Fig. 8E) and some of them expressed only one of them (Fig. 8, E and F). Thus, the overexpression of ACBP in COS-7 as determined by Western blotting represents the total amount of ACBP expressed in transfected cells but not necessarily ACBP simultaneously overexpressed with HNF-4 in the same cells.

View larger version (22K): [in this window] [in a new window]   FIG. 8. Indirect immunofluorescence (confocal) microscopy detection of ACBP and HNF-4 in COS-7 cells. Nontransfected (control) and transfected COS-7 cells were labeled with Texas Red and FITC so that ACBP was detected as green fluorescence and HNF-4 as red fluorescence. A, nontransfected (control) COS-7 cells; B, COS-7 cells transfected with pLEN4S (HNF-4 expression plasmid); C, COS-7 cells cotransfected with pLEN4S (HNF-4 expression vector) and pgkaACBP (ACBP antisense RNA expression vector); D, COS-7 cells transfected with pCI-sACBP (ACBP expression vector); E, COS-7 cells cotransfected with pLEN4S and pCI-sACBP, and the two proteins were expressed in same cell; F, COS-7 cells cotransfected with pLEN4S and pCI-sACBP, and the two proteins were expressed separately.

View larger version (46K): [in this window] [in a new window]   FIG. 9. Intracellular location of ACBP and HNF-4 in COS-7 cells that ectopically coexpressed the two proteins. A, ACBP (green fluorescence); B, HNF-4 (red fluorescence); C, DNA (conventionally blue fluorescence); D-F, green/blue (ACBP/DNA), red/blue (HNF-4/DNA), and green/red (ACBP/HNF-4) colocalization fluorographs, respectively. Fluorographs show the intensities and scatter pattern of all pixels within the image; pixels with mostly one fluorescent component are placed along the axis although the pixels with equal fluorescence intensity from both components (due to colocalization) are placed in between the axes, where the square regions of interest have been selected); the axes are green, blue, and red. F.I. measure green, blue, and red fluorescence intensity of the pixels on an arbitrary scale from 0 to 255 (according to LaserSharp software); G-I, images given by selected pixels (within the boxed areas in D-F) with highest colocalization of green/blue (ACBP/DNA), red/blue (HNF-4/DNA), and green/red (ACBP/HNF-4) fluorescence intensities, respectively.

View larger version (57K): [in this window] [in a new window]   FIG. 10. Intracellular location of ACBP and HNF-4 in rat hepatoma cells. A, ACBP (green fluorescence); B, HNF-4 (red fluorescence); C, DNA (conventionally blue fluorescence); D-F, green/blue (ACBP/DNA), red/blue (HNF-4/DNA), and green/red (ACBP/HNF-4) colocalization fluorographs, respectively. G-I, images of selected pixels with highest colocalization of green/blue (ACBP/DNA), red/blue (HNF-4/DNA), and green/red (ACBP/HNF-4) fluorescence intensities, respectively.

View larger version (23K): [in this window] [in a new window]   FIG. 11. Reporter assays indicate that ACBP stimulates HNF-4-mediated transactivation. Cells were transfected with reporter plasmid ApoBLuc and reference Renilla luciferase in all experiments. In addition, ACBP and HNF-4 expression vectors or empty vectors (for control) were cotransfected as indicated. A, luciferase transcription activity (relative light units) in COS-7 cells (bars 1-7) and hepatoma cells (bars 8-10) transfected with expression vectors as indicated. B, luciferase activity was determined in COS-7 cells transfected with 0.5 and 1.0 µg of sense ACBP expression vector (bars 5 and 6, respectively) or antisense ACBP plasmid (bars 9 and 10, respectively) in the presence of a constant amount of HNF-4 expression vector. Controls (bars 1-4 and 7 and 8) were run for cells transfected with no ACBP vector or empty vectors. s/a, sense/antisense.

A dose response of the ACBP expression versus luciferase activity was determined in COS-7 cells cotransfected with various amount of ACBP sense or antisense vector and constant amounts of HNF-4 expression plasmid and ApoBLuc reporter vector (Fig. 11B). Luciferase transactivation in COS-7 cells was directly proportional to transfection with increasing amounts of pCI-sACBP, the overexpression vector for ACBP (Fig. 11B, sense ACBP). Conversely, luciferase transactivation in COS-7 cells was inversely proportional to transfection with increasing amounts of antisense ACBP vector, pgk-aACBP (Fig. 11B, antisense-ACBP). Thus, ACBP stimulated HNF-4-mediated transactivation proportional to the level of ACBP expression in the COS-7 and rat hepatoma cells. The expression of either sense or antisense mRNAs for ACBP and HNF-4 proteins was strongly dependent on the cell type. Thus, pgk-aACBP promoter was a good antisense expression vector in COS-7 but not in rat hepatoma cells; in contrast, pLEN4S-HNF-4 was a good sense expression vector in COS-7 but not in hepatoma cells (data not shown). This differential efficiency of the same expression vector in different cell lines has been reported previously (36-38) for numerous promoters, in addition to the difference in transfection efficiency between COS-7 and hepatoma cells.

Functional Significance of ACBP Expression on HNF-4 Transcriptional Activity, LSCM Imaging of Luciferase Transactivation in Individual Cells—To assess more accurately the effect of ACBP expression on HNF-4-mediated luciferase transcription, a new technical approach was used. Transfected rat hepatoma cells were fixed and triple fluorescent-labeled to simultaneously detect HNF-4, ACBP, and luciferase. Thus, cells transfected with pCI-sACBP (ACBP overexpression vector) and ApoBLuc (luciferase reporter vector) were labeled with Texas Red for HNF-4, FITC for ACBP, and Cy5 for luciferase (Fig. 12, A-C). By image analysis, many individual cells were analyzed for their content in HNF-4, ACBP, and luciferase and then the correlation curves for the three protein expression levels were studied (Fig. 12, D and E). A plot of ACBP versus luciferase expression (Fig. 12D) revealed a very high correlation coefficient (r², 0.936) suggesting that cells with higher amounts of ACBP exhibited higher levels of luciferase (and implicitly had a higher HNF-4-mediated transactivation). As both luciferase versus HNF-4 and ACBP versus HNF-4 showed very good correlation (r², 0.899 and 0.856, respectively; plots not shown), luciferase and ACBP were normalized to HNF-4, and then the degree of correlation was again determined. Interestingly, for ratios of ACBP/HNF-4 lower than 0.7, a good correlation coefficient was found, i.e. r² of 0.853 (Fig. 12E). For ratios of ACBP/HNF-4 higher than 0.7, the correlation did not hold. As both ACBP and HNF-4 had been reported to bind fatty acyl-CoAs with very high affinities (in the low nanomolar range) (1, 17), the ratio between the two proteins may be a very important factor in the way ACBP influences HNF-4 activity.

View larger version (35K): [in this window] [in a new window]   FIG. 12. Confocal assay of luciferase transactivation in rat hepatoma cells demonstrates that ACBP stimulates HNF-4-mediated transactivation. Rat hepatoma cells transfected with ApoBLuc reporter plasmid and pCI-sACBP (ACBP expression vector) were labeled with Texas Red (red fluorescence, A) for HNF-4, FITC (green fluorescence, B) for ACBP and Cy5 (conventionally blue fluorescence, C) for luciferase, as described under "Experimental Procedures." D, correlation plot for luciferase (blue fluorescence intensity) and ACBP (green fluorescence intensity) expression. E, correlation plot of HNF-4-normalized ACBP fluorescence intensities versus HNF-4-normalized luciferase fluorescence intensities.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

One possibility is that by binding LCFA-CoAs with high affinity, the ACBP may compete with nuclear receptors for ligand binding and thereby regulate transcriptional activity of the nuclear receptors. Indeed LCFA-CoAs are present in the nuclei of rat liver cells (14) at physiologically significant levels, i.e. unbound LCFA-CoA are <10 nM (12, 16). LCFA-CoAs antagonize the effects of peroxisome proliferators on peroxisome proliferator-activated receptor PPAR- but not - or - (20). LCFA-CoAs modulate the transcriptional activity of HNF-4 in a ligand chain length and unsaturation-dependent manner (16, 18). Not only ACBP (5, 7) but also nuclear receptors such as HNF-4 (17, 18), TR (19), and PPAR- and - (20, 21) interact with LCFA-CoAs. The affinities of several of these proteins for LCFA-CoAs, especially ACBP and HNF-4, are in the physiological range of nuclear LCFA-CoA levels: ACBP (K_d of 0.6-4 nM) HNF-4 (K_d of 1.5-4nM) >> TR (K_d of 120 nM) >> PPAR (displaces Wy14643). Even though ACBP and HNF-4 both display very high affinities for LCFA-CoAs in vitro and might be competing for a common ligand, it is known that in other cases (e.g. carnityl-palmitoyltransferase) LCFA-CoAs bound to ACBP but not free LCFA-CoAs are preferentially taken further into a transport or metabolic pathway, e.g. mitochondrial oxidation (36). Interestingly, LCFAs bound to L-FABP but not free LCFAs are cotransported into nuclei (37).

Alternatively, ACBP may interact directly with nuclear transcription factors to influence transcriptional activity. Support for this possibility comes from the observation that ACBP expression significantly decreases PPAR--mediated transactivation (15). Since LCFA-CoAs antagonize the effects of peroxisome proliferators on PPAR- but not - or - (20), it is unlikely that the effects of ACBP on PPAR- are mediated through the ligand LCFA-CoAs. It has been shown that L-FABP interacts with PPAR- and - and stimulates fatty acid and hypolipidemic drug activation of PPAR-dependent transcription (38). Interestingly, Hertz et al. (39) demonstrated that overexpression of acyl-CoA synthase decreased the capacity of amphipathic carboxylic peroxisomal proliferators to induce PPAR-dependent transactivation, but it increased the effect of long chain fatty acids on HNF-4-dependent transactivation (16). This may suggest that free fatty acids interact readily with L-FABP·PPAR complexes, although acyl-CoAs affect primarily ACBP·HNF-4 complexes. The present work showed for the first time that there is a direct, physical, and functional interaction between ACBP and HNF-4.

First, in vitro studies by circular dichroism demonstrated that recombinant ACBP and HNF-4-ligand binding domain when mixed together interact to form a complex with altered conformation (Fig. 1). Altered conformation of nuclear receptors can modulate cofactor recruitment and thereby influence transcriptional activity (21). Circular dichroism has been used previously to detect profound changes in secondary structures of two proteins upon interaction to form a complex (40, 41). Thus, for example, by comparing the theoretical sum of individual far-UV circular dichroic spectra of estrogen receptor- and TATA box-binding protein to the actual spectrum of an equimolecular mixture of the two proteins, significant differences were obtained indicating that interaction between estrogen receptor- and TATA box-binding protein resulted in a conformational change in either or both proteins (40). Similarly, the present CD spectroscopy study demonstrates that a mixture of HNF-4-LBD and ACBP recombinant proteins exhibited a CD spectrum that was different from the theoretical sum of individual protein CD spectra, indicating a structural change upon HNF-4-LBD complex formation with ACBP (Fig. 1 and Table I). It is well known that nuclear receptors when purified as recombinant proteins exhibit a partially unstructured or random-coiled conformation in buffered aqueous solutions in contrast with their conformation in x-ray crystals (42). Hydrophobic solvents like trifluoroethanol or specific proteins acting as coactivators can increase the helical structure of nuclear receptor molecules in buffered aqueous solutions (40, 42). Under our experimental conditions, in aqueous buffer HNF-4-LBD had a low helical structure; in contrast, ACBP, a very soluble protein in aqueous buffer, had a high content of -helix, but in the presence of HNF-4-LBD it formed a complex with a CD spectrum indicating similarity to the individual HNF-4-LBD but with a higher content of -helix than HNF-4-LBD alone (Fig. 1 and Table I).

Second, ACBP directly interacted with HNF-4 in hepatoma cells in vitro and liver tissue in vivo as demonstrated by coimmunoprecipitation, immunogold electron microscopy, and confocal microscopy. HNF-4 was detected in anti-ACBP immunoprecipitates of liver and hepatoma cell homogenates (Fig. 2) and also by immunogold electron microscopy in nuclei of mouse liver cells (Fig. 3). Control experiments for the specificity of ACBP/HNF-4 association were run for both techniques used. In coimmunoprecipitation experiments, ACBP was found in precipitates generated with anti-HNF-4 but not with anti-Sp1 antibody. Immunoprecipitates produced with anti-ACBP antibody contained HNF-4 but not Sp-1 protein or GR nuclear receptor. In the literature, direct physical interaction of Sp1 protein with numerous nuclear transcription factors such as RAR/RXR (43), TR, vitamin D₃ receptor, and PPAR have been reported (44). Regarding HNF-4, however, the literature is controversial. Even though several reports (45-48) demonstrated close proximity of response elements recognized by HNF-4 and Sp1 within regulatory sequences of several genes, only one article (49) suggested a direct interaction of HNF-4 with the Sp1 protein. The latter results are different from our immunoprecipitation data that showed no association of ACBP and Sp1 protein in mouse liver and rat hepatoma cells and may be explained by the use of different experimental conditions. For example, the pull-down assays described in this article (49) were run with in vitro synthesized ³⁵S-labeled HNF-4 and GST-Sp1 fusion proteins, which might have favored finding protein-protein association because of the following reasons. (i) In vitro incubation of large amounts of purified proteins may conduct nonspecific binding between them. (ii) ³⁵S-Labeled protein detection is severalfold more sensitive than the colorimetric (alkaline-phosphatase/colorimetric substrate) or chemiluminescent (horseradish peroxidase/chemiluminescent substrate) methods that were used in our experiments. In the same report (49), coimmunoprecipitation of FLAG-HNF-4 with Sp1 was demonstrated only in HepG2 cells that had been transfected to overexpress FLAG-tagged HNF-4 but not in nontransfected cells. This experimental approach does not exclude a nonspecific association between overexpressed FLAG-HNF-4 and Sp1. The fact that in our experiments, ACBP was found associated with HNF-4 but not with Sp1 suggested that either HNF-4 was not physically connected to Sp1 at all times under the experimental conditions employed or that ACBP was associated with HNF-4 that was not involved with Sp1. Using immunogold electron microscopy as a technique that provides accurate detection of protein-protein interactions, ACBP was found closely associated with HNF-4 (within 43 Å) but not with GR. Confocal microscopy experiments confirmed that ACBP and HNF-4 significantly colocalized in the nuclei of rat hepatoma (nontransfected cells) and COS-7 cells transfected with expression vectors for ACBP and HNF-4 (Figs. 4, 9, and 10). The intermolecular distance between ACBP and HNF-4 was determined to be 53 Å by FRET microscopy (Fig. 5). Additional support for close molecular interaction between these proteins was provided by mammalian two-hybrid assay with ACBP and HNF-4 cDNAs ligated to pACT and pBIND (Fig. 6).

Third, transactivation assays demonstrated that ACBP interaction with HNF-4 was functionally significant. Functional studies in COS-7 cells cotransfected with HNF-4, ACBP expression vectors, and ApoBLuc reporter plasmid indicated that ACBP had a stimulatory effect on HNF-4-mediated transactivation (Fig. 11A). An overexpression of ACBP in rat hepatoma cells resulted in a significant increase in HNF-4-mediated transactivation (Fig. 11A). In COS-7 cells, a dose response in HNF-4-mediated transactivation over a range of ACBP sense and antisense expression vectors was obtained, demonstrating that the more ACBP was expressed in the cells the higher the HNF-4-mediated transactivation was observed (Fig. 11B). When individual cells were examined for the expression of ACBP, HNF-4, and luciferase reporter, correlation curves demonstrated that individual cells with higher amounts of ACBP expressed more luciferase (Fig. 12). These data suggest that the stimulatory effect of ACBP on HNF-4-mediated transactivation could be explained by the ability of ACBP to interact directly with HNF-4 to alter its conformation. Interestingly, the stimulatory effect of ACBP on HNF-4 transactivation function is in agreement with ACBP inhibitory effects on PPAR (15, 20). Cross-talk between HNF-4 and PPAR has been demonstrated to regulate apoC-III gene through common DR-1 consensus elements in its promoter (50). Thus, the overall process may include apoC-III transcription stimulation when ACBP (or ACBP/LCFA-CoA) activates HNF-4 to bind to DR-1-response elements, in balance with the opposite effect, i.e. apoC-III transcription down-regulation when free fatty acid/LFABP stimulates PPAR/retinoid X receptor heterodimers to displace HNF-4 homodimers from DR-1 cis-elements.

In summary, the data presented herein demonstrate for the first time that ACBP directly interacts with HNF-4 in vitro,in intact fixed cells, and in living cells. Furthermore, ACBP expression levels directly correlated with HNF-4-mediated transactivation in transfected cells. These data were consistent with ACBP directly affecting HNF-4 transcriptional activity and/or altering the nuclear distribution of LCFA-CoA ligands that activate or inhibit HNF-4 (16-18). Based on the fact that LCFA is cotransported to the nucleus as an LCFA·L-FABP complex and not as free LCFA (37), a possible scenario is that LCFA-CoA·ACBP complexes may also pass through the nuclear membrane pores.

Finally, the fact that ACBP directly interacts with HNF-4 to alter its structure provides for the first time a novel mechanism, possibly independent of specific ligands, whereby a primarily cytoplasmic protein involved in LCFA-CoA metabolism may affect the transcriptional activity of a nuclear regulatory protein. Regardless of whether HNF-4 binds no ligand, binds LCFA-CoA, and/or binds LCFA, the present data demonstrate the physical and functional interaction of ACBP with HNF-4. This suggests for the first time that ACBP may act as a coregulator involved in the transcription of genes targeted by HNF-4. How this interaction relates to LCFA-CoA interaction with HNF-4 is beyond the scope of the present investigation but is an interesting future possibility to be examined.

	FOOTNOTES

 
^* This work was supported in part by United States Public Health Service Grant DK41402 (to F. S.) and the NIEHS Center for Rural and Environmental Health Grant ES09106 from the National Institutes of Health (to A. B. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^|| To whom correspondence should be addressed: Dept. of Pathobiology, Texas A & M University, Texas Veterinary Medical Center, College Station, TX 77843-4467. Tel.: 979-862-1509; Fax: 979-845-9231; E-mail: Akier{at}cvm.tamu.edu.

¹ The abbreviations used are: ACBP, acyl-CoA-binding protein; FRET, fluorescence resonance energy transfer; GR, glucocorticoid receptor; HNF-4, hepatocyte nuclear factor 4; HNF-4-LBD, hepatocyte nuclear factor 4-ligand binding domain (amino acids 132-455); LCFA, long chain fatty acid; LCFA-CoA, long chain fatty acyl-CoA; LSCM, laser scanning confocal microscopy; PPAR, peroxisome proliferator-activated receptor; TR, thyroid receptor; FITC, fluorescein isothiocyanate; L-FABP, liver-fatty acid-binding protein.

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