Horng-Yuan Kan

The Central Helices of ApoA-I Can Promote ATP-binding Cassette Transporter A1 (ABCA1)-mediated Lipid Efflux AMINO ACID RESIDUES 220–231 OF THE WILD-TYPE ApoA-I ARE REQUIRED FOR LIPID EFFLUX IN VITRO AND HIGH DENSITY LIPOPROTEIN FORMATION IN VIVO

We have mapped the domains of lipid-free apoA-I that promote cAMP-dependent and cAMP-independent cholesterol and phospholipid efflux. The cAMP-dependent lipid efflux in J774 mouse macrophages was decreased by ∼80–92% by apoA-I[Δ(185–243)], only by 15% by apoA-I[Δ(1–41)] or apoA-I[Δ(1–59)], and was restored to 75–80% of the wild-type apoA-I control value by double deletion mutants apoA-I[Δ(1–41)Δ(185–243)] and apoA-I[Δ(1–59)Δ(185–243)]. Similar results were obtained in HEK293 cells transfected with an ATP-binding cassette transporter A1 (ABCA1) expression plasmid. The double deletion mutant of apoA-I had reduced thermal and chemical stability compared with wild-type apoA-I. Sequential carboxyl-terminal deletions showed that cAMP-dependent cholesterol efflux was diminished in all the mutants tested, except the apoA-I[Δ(232–243)] which had normal cholesterol efflux. In cAMP-untreated or in mock-transfected cells, cholesterol efflux was not affected by the amino-terminal deletions, but decreased by 30–40% and 50–65% by the carboxyl-terminal and double deletions, respectively. After adenovirus-mediated gene transfer in apoA-I-deficient mice, wild-type apoA-I and apoA-I[Δ(1–41)] formed spherical high density lipoprotein (HDL) particles, whereas apoA-I[Δ(1–41)Δ(185–243)] formed discoidal HDL. The findings suggest that although the central helices of apoA-I alone can promote ABCA1-mediated lipid efflux, residues 220–231 are necessary to allow functional interactions between the full-length apoA-I and ABCA1 that are required for lipid efflux and HDL biogenesis.

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

The transcription of eukaryotic genes is controlled by the interaction of regulatory gene sequences (promoter elements) with specific nuclear proteins (transcription factors) (1–3). The interaction of the transcription factors with the promoter elements controls: a) tissue specific gene expression (4–6); b) gene expression during differentiation and development (7,8); and c) gene expression in response to intracellular and extracellular stimuli such as hormones and metabolites (9–12).

Probing the pathways of chylomicron and HDL metabolism using adenovirus-mediated gene transfer

Purpose of the review This review clarifies the functions of key proteins of the chylomicron and the HDL pathways. Recent findings Adenovirus-mediated gene transfer of several apolipoprotein (apo)E forms in mice showed that the amino-terminal 1-185 domain of apoE can direct receptor-mediated lipoprotein clearance in vivo. Clearance is mediated mainly by the LDL receptor. The carboxyl-terminal 261-299 domain of apoE induces hypertriglyceridemia, because of increased VLDL secretion, diminished lipolysis and inefficient VLDL clearance. Truncated apoE forms, including apoE2-202, have a dominant effect in remnant clearance and may have future therapeutic applications for the correction of remnant removal disorders. Permanent expression of apoE and apoA-I following adenoviral gene transfer protected mice from atherosclerosis. Functional assays, protein cross-linking, and adenovirus-mediated gene transfer of apoA-I mutants in apoA-I deficient mice showed that residues 220-231, as well as the central helices of apoA-I, participate in ATP-binding cassette transporter A1-mediated lipid efflux and HDL biogenesis. Following apoA-I gene transfer, an amino-terminal deletion mutant formed spherical α-HDL, a double amino- and carboxyl-terminal deletion mutant formed discoidal HDL, and a carboxyl-terminal deletion mutant formed only pre-β-HDL. The findings support a model of cholesterol efflux that requires direct physical interactions between apoA-I and ATP-binding cassette transporter A1, and can explain Tangier disease and other HDL deficiencies. Summary New insights are provided into the role of apoE in cholesterol and triglyceride homeostasis, and of apoA-I in the biogenesis of HDL. Clearance of the lipoprotein remnants and increase in HDL synthesis are obvious targets for therapeutic interventions.

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.

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-Igene, 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 apoCIIIenhancer 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 andapoCIII 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-Igene 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.

Regulatory gene mutations affecting apolipoprotein gene expression: functions and regulatory behavior of known genes may guide future pharmacogenomic approaches to therapy.

Abstract A pharmacogenomic approach to therapy requires systematic knowledge of the regulatory regions of the genes, as well as basic understanding of transcriptional regulatory mechanisms of genes. Using the apolipoprotein (apo) A-I/CIII gene cluster as a model system, we have identified by in vitro and in vivo studies the regulatory elements and the factors which control its transcription. Studies in transgenic mice established that the hepatocyte nuclear factor (HNF-4) binding site of the apoCIII enhancer, which controls transcription of both genes, is required for the intestinal expression of apoA-I and apoCIII genes, and enhances synergistically their hepatic transcription 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. The regulation of the apoE/apoCI/apoCIV/apoCII cluster is also cited. It is expected that identification of the regulatory regions of genes will be soon accelerated by the sequencing of several mammalian genomes. The functional analyses of the regulatory domains of genes involved in lipid homeostasis, combined with cross-species sequence comparisons in the near future, may identify natural regulatory gene polymorphisms in the general population that will permit rational pharmacogenomic approaches for treatment of dyslipidemias.