In vivo studies of HDL assembly and metabolism using adenovirus-mediated transfer of ApoA-I mutants in ApoA-I-deficient mice.

We have used adenovirus-mediated gene transfer in apoA-I-deficient (A-I-/-) mice to probe the in vivo assembly and metabolism of HDL using apoA-I variants, focusing primarily on the role of the C-terminal 32 amino acids (helices 9-10). Lipid, lipoprotein, and apoA-I analyses showed that plasma levels of apoA-I and HDL of the mutants were 40-88% lower than that of wild type (WT) human apoA-I despite comparable levels of expression in the liver. WT apoA-I and mutant 1 (P165A, E172A) formed spherical particles with the size and density of HDL2 and HDL3. Mutant 2 (E234A, E235A, K238A, K239A) generated spherical particles with density between HDL2 and HDL3. Mutant 3 (L211V, L214V, L218V, L219V) and mutant 4 (L222K, F225K, F229K), which have substitutions of hydrophobic residues in the C-terminus, generated discoidal HDL particles indicating a defect in their conversion to mature spherical HDL. Significant amounts of mutant 4 and mutant 5 (truncated at residue 219) were found in the lipid poor fractions after ultracentrifugation of the plasma (18 and 35%, respectively, of total apoA-I). These findings suggest that hydrophobic residues in and/or between helices 9 and 10 are important for the maturation of HDL in vivo.

Transcriptional regulatory mechanisms of the human apolipoprotein genes in vitro and in vivo.

The present review summarizes recent advances in the transcriptional regulation of the human apolipoprotein genes, focusing mostly, but not exclusively, on in-vivo studies and signaling mechanisms that affect apolipoprotein gene transcription. An attempt is made to explain how interactions of transcription factors that bind to proximal promoters and distal enhancers may bring about gene transcription. The experimental approaches used and the transcriptional regulatory mechanisms that emerge from these studies may also be applicable in other gene systems that are associated with human disease. Understanding extracellular stimuli and the specific mechanisms that underlie apolipoprotein gene transcription may in the long run allow us to selectively switch on antiatherogenic genes, and switch off proatherogenic genes. This may have beneficial effects and may confer protection from atherosclerosis to humans.

Transcriptional regulation of the human apolipoprotein genes.

This review provides experiments and putative mechanisms which underlie the transcription of the human apolipoprotein genes in vitro and in vivo. Summarized below are the key findings for individual genes and gene clusters. ApoA-II. 1- The -911/+29 promoter is sufficient to direct expression of a reporter gene exclusively in the liver and thus represents a liver-specific promoter. 2- Important factors for the activity of this promoter are hormone nuclear receptors and the ubiquitous factor USF. 3. SREBP-1 and SREBP-2 bind to five and four sites respectively and transactivate the apoA-II promoter. Their role in the in vivo transcription of the apoA-II gene has not been established. ApoB. 1. Regulatory sequence extending 5 Kb upstream and 1.5 Kb downstream of the apoB promoter are sufficient to direct hepatic expression of the apoB gene. The intestinal expression of the apoB gene requires in addition a 315 bp intestinal enhancer located 56 Kb upstream of the apoB gene. 2. Important factors for apoB gene transcription appear to be C/EBP, HNF-3, HNF-4 and other nuclear receptors which bind both on the proximal promoter and the intestinal enhancer. ApoE/ApoCI/ApoCIV/ApoCII Cluster. 1. The expression of the genes of the apoE/apoCI/apoCII/apoE cluster are controlled by two homologous hepatic control regions designated HCR-1 and HCR-2 of approximately 600 bp located 15 and 27 Kb 3? of the apoE gene. Either region is sufficient to direct gene expression in vivo, although HCR-1 appears to have a dominant effect on apoE and apoCI and HCR-2 has a dominant effect on apoCIV and apoCII gene expression. 2. Two other homologous regulatory regions designated ME-1 and ME-2 located 3.3 and 15.9 Kb downstream of the apoE gene can direct independently the expression of the apoE gene in macrophages and adipocytes. 3. Important factors for apoE gene regulation appear to be SP1 on the proximal promoter, and possibly HNF-3, C/EBP and hormone nuclear receptors on the enhancers. 4. Important factors for apoCII gene transcription appear to be HNF-4 and RXR-alpha/T3R-beta which binds to a thyroid response element of the proximal promoter. ApoA-I/ApoCIII/ApoA-IV Gene Cluster. 1. The transcription of the apoA-I/apoCIII/apoA-IV gene cluster is controlled by a common enhancer located 590 to 790 nucleotides upstream of the apoCIII gene. 2. Important factors for the activity of the enhancer are SP1, HNF-4 and possibly other nuclear receptors. Important factors for the activity of the proximal promoters are HNF-4, and possibly other nuclear receptors. 3. The HNF-4 binding site of the apoCIII enhancer is required for the intestinal expression of apoA-I and apoCIII gene and enhances synergistically the hepatic transcription of the two genes and possibly of apoA-IV in vivo. The three SP1 sites of the enhancer are also required for the intestinal expression of apoA-I and apoCIII genes in vivo and for the enhancement of the hepatic transcription. 4. Pro-inflammatory cytokines such as TNF-alpha and IL-1 repress, and TGF-beta stimulates the apoCIII promoter activity. The TGF-beta pathway activates SMAD3/4 proteins which interact with HNF-4 bound to the apoCIII promoter and enhancer and increase its activity. 5. It appears that other factors activated by different signaling pathways (NF-kappa-B, Jun and others) interact with HNF-4 bound to the enhancer and thus repress the activity of apoCIII promoter. Understanding the transcriptional regulatory mechanism of the apolipoprotein genes may allow, in the long run, selective increase of anti-atherogenic lipoproteins and thus reduce the risk of cardiovascular disease.

A hormone response element in the human apolipoprotein CIII (ApoCIII) enhancer is essential for intestinal expression of the ApoA-I and ApoCIII genes and contributes to the hepatic expression of the two linked genes in transgenic mice.

We have generated transgenic mice carrying wild-type promoters of the human apolipoprotein A-I (apoA-I)-apoCIII gene cluster or promoters mutated in their hormone response elements. The wild-type cluster directed high levels of apoA-I gene expression in liver and intestine, moderate expression in kidney, and low to minimal expression in other tissues. It also directed high levels of chloramphenicol acetyltransferase (CAT) expression (used as a reporter for the apoCIII gene) in liver, low levels in intestine and kidney, and no expression in other tissues. Mutations in the apoCIII promoter and enhancer abolished the intestinal and renal expression of the apoA-I gene, reduced hepatic apoA-I expression by 80%, and abolished CAT expression in all tissues. A similar pattern of expression was obtained by mutations in the apoCIII enhancer alone. Mutations in the proximal apoA-I promoter reduced by 85% hepatic and intestinal apoA-I expression and did not affect CAT expression. The findings suggest that a hormone response element within the apoCIII enhancer is essential for intestinal and renal expression of apoA-I and apoCIII genes and also enhances hepatic expression. The hormone response elements of the proximal apoA-I promoter or the apoCIII enhancer can promote independently low levels of hepatic and intestinal expression of the apoA-I gene in vivo.

The SP1 sites of the human apoCIII enhancer are essential for the expression of the apoCIII gene and contribute to the hepatic and intestinal expression of the apoA-I gene in transgenic mice.

We have generated transgenic mice carrying wild-type and mutant forms of the apolipoprotein (apo)A-I/apoCIII gene cluster. Mutations were introduced either in one or in three SP1 binding sites of the apoCIII enhancer. In mice carrying the wild-type transgene, major sites of apoA-I mRNA synthesis were liver and intestine and minor sites were kidney and, to a lesser extent, other tissues. The major site of chloramphenicol acetyl transferase (CAT) activity (used as a reporter for the apoCIII gene) was liver and minor sites intestine and kidney. A mutation in one SP1 binding site reduced the expression of the apoA-I gene to approximately 23 and 19% in the liver and intestine, respectively, as compared to the control wild-type. The hepatic expression of the CAT gene was not affected whereas the intestinal expression was nearly abolished. Mutations in three SP1 binding sites reduced the hepatic and intestinal expression of the apoA-I and CAT genes to 14 and 4%, respectively, as compared to the wild-type control, and abolished CAT expression in all tissues. The findings suggest that the SP1 sites of the apoCIII enhancer are required for the expression of the apoCIII gene and also contribute significantly to the hepatic and intestinal expression of the apoA-I gene in vivo.

SREBP-1 binds to multiple sites and transactivates the human ApoA-II promoter in vitro : SREBP-1 mutants defective in DNA binding or transcriptional activation repress ApoA-II promoter activity.

-Screening of an expression human liver cDNA library resulted in the isolation of several cDNA clones homologous to sterol regulatory element-binding protein-1 (SREBP-1) that recognize the regulatory element AIIAB and AIIK of the human apoA-II promoter. DNaseI footprinting of the apoA-II promoter using SREBP-1 (1 to 460) expressed in bacteria identified 5 overall protected regions designated AIIAB (-64 to -48), AIICD (-178 to -154), AIIDE (-352 to -332), AIIHI (-594 to -574), and AIIK (-760 to -743). These regions contain inverted E-box palindromic or direct repeat motifs and bind SREBP-1 with different affinities. Transient cotransfection experiments in HepG2 cells showed that SREBP-1 transactivated the -911/29 apoA-II promoter 3.5-fold as well as truncated apoA-II promoter segments that contain 1, 2, 3, or 4 SREBP binding sites. Mutagenesis analysis showed that transactivation by SREBP was mainly affected by mutations in element AIIAB. Despite the strong transactivation of the apoA-II promoter by SREBP-1 we could not demonstrate significant changes on the endogenous apoA-II mRNA levels of HepG2 cells after cotransfection with SREBP-1 or in the presence or absence of cholesterol and 25-OH-cholesterol. An SREBP-1 mutant lacking the amino-terminal activation domain bound normally to its cognate sites and repressed the apoA-II promoter activity. Repression was also caused by specific amino acid substitutions of Leu, Val, or Gly for Lys359, which affected DNA binding. Repression by the DNA binding-deficient mutants was abolished by deletion of the amino-terminal activation domain (1 to 90) of SREBP-1. Overall, the findings suggest that the wild-type SREBP-1 can bind and transactivate efficiently the apoA-II promoter in cell culture. SREBP-1 mutants lacking the activation domain bind to their cognate sites and directly repress the apoA-II promoter whereas mutants defective in DNA binding indirectly repress the apoA-II promoter activity, possibly by a squelching mechanism.

The -700/-310 fragment of the apolipoprotein A-IV gene combined with the -890/-500 apolipoprotein C-III enhancer is sufficient to direct a pattern of gene expression similar to that for the endogenous apolipoprotein A-IV gene.

Spatial gene expression in the intestine is mediated by specific regulatory sequences. The three genes of the apoA-I/C-III/A-IV cluster are expressed in the intestine following cephalocaudal and crypt-to-villus axes. Previous studies have shown that the -780/-520 enhancer region of the apoC-III gene directs the expression of the apoA-I gene in both small intestinal villi and crypts, implying that other unidentified elements are necessary for a normal intestinal pattern of apoA-I gene expression. In this study, we have characterized transgenic mice expressing the chloramphenicol acetyltransferase gene under the control of different regions of the apoC-III and apoA-IV promoters. We found that the -890/+24 apoC-III promoter directed the expression of the reporter gene in crypts and villi and did not follow a cephalocaudal gradient of expression. In contrast, the -700/+10 apoA-IV promoter linked to the -500/-890 apoC-III enhancer directed the expression of the reporter gene in enterocytes with a pattern of expression similar to that of the endogenous apoA-IV gene. Furthermore, linkage of the -700/-310 apoA-IV distal promoter region to the -890/+24 apoC-III promoter was sufficient to restore the appropriate pattern of intestinal expression of the reporter gene. These findings demonstrate that the -700/-310 distal region of the apoA-IV promoter contains regulatory elements that, in combination with proximal promoter elements and the -500/-890 enhancer, are necessary and sufficient to restrict apoC-III and apoA-IV gene expression to villus enterocytes of the small intestine along the cephalocaudal axis.

DNA binding specificity and transactivation properties of SREBP-2 bound to multiple sites on the human apoA-II promoter.

DNase I footprinting of the apoA-II promoter using sterol regulatory element binding protein-2 [(SREBP-2 (1-458)] expressed in bacteria identified four protected regions, designated AIIAB (-64 to -48), AIICD (-178 to -154), AIIDE (-352 to -332) and AIIK (-760 to -743), which bind SREBP-2 and contain either palindromic or direct repeat motifs. Potassium permanganate and dimethyl sulfate interference experiments using the AIIAB region as probe showed that the nucleotides of a decameric palindromic repeat RTCAMVTGMY and two 5' T residues participate in DNA-protein interactions. SREBP-2 transactivated the intact (-911/+29) apoA-II promoter 1.7-fold and truncated apoA-II promoter segments which contain one, two or three SREBP-2 sites 11- to 17-fold in HepG2 cells. Transactivation of a promoter construct containing the binding site AIIAB and the apoA-II enhancer, which includes the binding site AIIK, was abolished by mutations in element AIIAB. An SREBP-2 mutant defective in DNA binding caused a dose-dependent repression of the apoA-II promoter activity. Repression was also caused by an SREBP-2 mutant which lacks the N-terminal activation domain (residues 1-93) but binds normally to its cognate sites. In contrast, a double SREBP-2 mutant which lacks both the DNA binding and the activation domains has no effect on the apoA-II promoter activity. Overall, the findings suggest that SREBP-2 can transactivate the apoA-II promoter by binding to multiple sites. Furthermore, the repression caused by the DNA binding deficient mutants results from squelching of positive activator(s) which appear to recognize the activation domain of SREBP-2.