Vassilis I. Zannis

SMAD Proteins Transactivate the Human ApoCIII Promoter by Interacting Physically and Functionally with Hepatocyte Nuclear Factor 4

Cotransfection of HepG2 cells with SMADs established that SMAD3 and SMAD3-SMAD4 transactivated (15–70-fold) the −890/+24 apoCIII promoter and shorter promoter segments, whereas cotransfection with a dominant negative SMAD4 mutant repressed the apoCIII promoter activity by 50%, suggesting that SMAD proteins participate in apoCIII gene regulation. Transactivation required the presence of a hormone response element, despite the fact that SMADs could not bind directly to it. Cotransfection of SMAD3-SMAD4 along with hepatocyte nuclear factor-4 resulted in a strong synergistic transactivation of the −890/+24 apoCIII promoter, proximal promoter segments, or synthetic promoters containing either the apoCIII enhancer or the proximal apoCIII hormone response element. Inhibition of endogenous hepatocyte nuclear factor-4 synthesis by an antisense ribozyme construct reduced the constitutive activity of the apoCIII promoter in HepG2 cells to 10% and abolished the SMAD-mediated transactivation. Co-immunoprecipitation and GST pull-down assays provided evidence for physical interactions between SMAD3, SMAD4, and hepatic nuclear factor-4. Our findings indicate that transforming growth factor β and its signal transducer SMAD proteins can modulate gene transcription by novel mechanisms that involve their physical and functional interaction with hepatocyte nuclear factor-4, suggesting that SMAD proteins may play an important role in apolipoprotein gene expression and lipoprotein metabolism.

Isolation and characterization of a third isoform of human hepatocyte nuclear factor 4

Hepatocyte nuclear factor 4 (HNF-4) is an essential positive regulator of a large number of liver-specific genes. We report here the isolation of three HNF-4 isoforms from a human liver cDNA library. hHNF-4A and hHNF-4B, differing by the insertion of 10 amino acids in the C-terminal region, have been previously identified in mouse, rat and human liver. The novel isoform, hHNF-4C, is identical to hHNF-4A and B in the regions encompassing the DNA-binding and dimerization domains, but contains a different C-terminal domain. Similar to the other isoforms, hHNF-4C is produced in a limited number of tissues and represents 2.6-13% of the total hHNF-4 mRNA population, depending on the cell type. The chromosomal origin of all three isoforms has been localized to human chromosome 20. hHNF-4C can form heterodimers with hHNF-4A and B in vitro, and exhibits similar transactivation potential as hHNF-4A or B in transient transfection assays, suggesting that the divergent C-terminal region is not part of the transactivation domain.

Distal Apolipoprotein C-III Regulatory Elements F to J Act as a General Modular Enhancer for Proximal Promoters That Contain Hormone Response Elements Synergism Between Hepatic Nuclear Factor-4 Molecules Bound to the Proximal Promoter and Distal Enhancer Sites

Transient transfection assays have shown that the distal apoC-III promoter segments that contain the regulatory elements F to J enhance the strength of the tandemly linked proximal apoA-I promoter 5- to 13-fold in hepatic (HepG2) cells. Activation in intestinal (CaCo-2) cells to levels comparable to those obtained in HepG2 cells requires a larger apoA-I promoter sequence that extends to nucleotide -1500 as well as the presence of hepatic nuclear factor-4 (HNF-4). The distal apoC-III regulatory elements can also enhance 4- to 8-fold the strength of the heterologous apoB promoter in HepG2 and CaCo-2 cells. Finally, these elements in the presence of HNF-4 enhance 14.5- to 18.5-fold the strength of the minimal adenovirus major late promoter linked to two copies of the hormone response element (HRE) AID of apoA-I in both HepG2 and CaCo-2 cells. In vitro mutagenesis of the promoter/enhancer cluster established that the enhancer activity is lost by a mutation in the HRE present in the 3 end of the regulatory element I (-736 to -714) and is reduced significantly by point mutations or deletions in one or more of the regulatory elements F to J of the apoC-III enhancer. The enhancer activity also requires the HREs of the proximal apoA-I promoter. The apoC-III enhancer can also restore the activity of the proximal apoA-I and apoB promoters that have been inactivated by mutations in CCAAT/enhancers binding protein binding sites, indicating that C/EBP may not participate in the synergistic activation of the promoter/enhancer cluster. The findings suggest that the regulatory elements F to J of the apoC-III promoter act as a general modular enhancer that can potentiate the strength of proximal promoters that contain HREs. Such potentiation in the HepG2 cells can be accounted for by synergistic interactions between HNF-4 or other nuclear hormone receptors bound to the proximal and distal HREs and SP1 or other factors bound to the apoC-III enhancer. Additional factors may be required for optimal activity in CaCo-2 cells as well as for the function of this region as an intestinal enhancer.

Transcriptional Regulation of the Genes Involved in Lipoprotein Transport The Role of Proximal Promoters and Long-range Regulatory Elements and Factors in Apolipoprotein Gene Regulation

The transcription of eukaryotic genes is a complex biological event involving numerous proteins—including RNA polymerase II, the proteins of the basal transcription initiation complex, and a variety of promoter- and enhancer-specific transcription factors—and requiring an ATP-dependent activation step.1 2 3 4 5 6 7 8 9 10 11 12 The regulation of transcription is responsible for the tissue-specific gene expression as well as gene expression during differentiation and development and in response to intracellular and extracellular stimuli such as hormones and metabolites.nnNumerous studies have established that a precise array of regulatory elements exists in each promoter/enhancer and these elements are occupied by transcription factors. It has been proposed that this promoter/enhancer-specific arrangement of factors permits the formation of stereospecific DNA-protein complexes. These complexes may directly or indirectly interact with the basal transcription system, thus leading to the transcriptional activation of the target gene.8 13 nnSeveral experimental advances have facilitated the study of eukaryotic promoters and have led to the identification and characterization of several eukaryotic transcription factors. These include the following: (1) Definition of the long-range regulatory elements that confer tissue specificity or developmentally regulated expression. This analysis utilizes transgenic mouse technologies.14 15 (2) Definition of the promoter region a few kilobases upstream of the transcription initiation site necessary for gene transcription. This analysis monitors the expression of a reporter gene under the control of normal and mutated promoters after transfection of cell cultures. (3) Identification of the different factors that bind to a specific promoter region and definition of their binding sites on the DNA. For this purpose, several techniques are used, including DNase I footprinting, in vivo footprinting,16 17 18 gel electrophoretic mobility shift assays,19 supershift assays, and DNA binding interference assays that involve modification of T residues by KMnO4 and …

Inhibition of hepatocyte nuclear factor 4 transcriptional activity by the nuclear factor κB pathway

HNF-4 (hepatocyte nuclear factor 4) is a key regulator of liver-specific gene expression in mammals. We have shown previously that the activity of the human APOC3 (apolipoprotein C-III) promoter is positively regulated by the anti-inflammatory cytokine TGFbeta (transforming growth factor beta) and its effectors Smad3 (similar to mothers against decapentaplegic 3) and Smad4 proteins via physical and functional interactions between Smads and HNF-4. We now show that the pro-inflammatory cytokine TNFalpha (tumour necrosis factor alpha) antagonizes TGFbeta for the regulation of APOC3 gene expression in hepatocytes. TNFalpha was a strong inhibitor of the activity of apolipoprotein promoters that harbour HNF-4 binding sites and this inhibition required HNF-4. Using specific inhibitors of TNFalpha-induced signalling pathways, it was shown that inhibition of the APOC3 promoter by TNFalpha involved NF-kappaB (nuclear factor kappaB). Latent membrane protein 1 of the Epstein-Barr virus, which is an established potent activator of NF-kappaB as well as wild-type forms of various NF-kappaB signalling mediators, also inhibited strongly the APOC3 promoter and the transactivation function of HNF-4. TNFalpha had no effect on the stability or the nuclear localization of HNF-4 in HepG2 cells, but inhibited the binding of HNF-4 to the proximal APOC3 HRE (hormone response element). Using the yeast-transactivator-GAL4 system, we showed that both AF-1 and AF-2 (activation functions 1 and 2) of HNF-4 are inhibited by TNFalpha and that this inhibition was abolished by overexpression of different HNF-4 co-activators, including PGC-1 (peroxisome-proliferator-activated-receptor-gamma co-activator 1), CBP [CREB (cAMP-response-element-binding protein) binding protein] and SRC3 (steroid receptor co-activator 3). In summary, our findings indicate that TNFalpha, or other factors that trigger an NF-kappaB response in hepatic cells, inhibit the transcriptional activity of the APOC3 and other HNF-4-dependent promoters and that this inhibition could be accounted for by a decrease in DNA binding and the down-regulation of the transactivation potential of the AF-1 and AF-2 domains of HNF-4.

Transactivation of the Human Apolipoprotein CII Promoter by Orphan and Ligand-dependent Nuclear Receptors THE REGULATORY ELEMENT CIIC IS A THYROID HORMONE RESPONSE ELEMENT

The regulatory elements CIIC (−159/−116) and CIIB (−102/−81) of the apolipoprotein CII (apoCII) promoter have distinct specificities for orphan nuclear receptors (Vorgia, P., Zannis, V. I., and Kardassis, D. (1998) J. Biol. Chem. 273, 4188–4199). In this communication we investigated the contribution of ligand-dependent and orphan nuclear receptors on the transcriptional regulation of the humanapoCII gene. It was found that element CIIC in addition to ARP-1 and EAR-2 binds RXRα/T3Rβ heterodimers strongly, whereas element CIIB binds hepatic nuclear factor 4 (HNF-4) exclusively. Binding is abolished by mutations that alter the HRE binding motifs. Transient cotransfection experiments showed that in the presence of T3, RXRα/T3Rβ heterodimers transactivated the −205/+18 apoCII promoter 1.6- and 11-fold in HepG2 and COS-1 respectively. No transactivation was observed in the presence of 9-cis-retinoic acid. Transactivation requires the regulatory element CIIC, suggesting that this element contains a thyroid hormone response element. HNF-4 did not affect the apoCII promoter activity in HepG2 cells. However, mutations in the HNF-4 binding site on element CIIB and inhibition of HNF-4 synthesis in HepG2 cells by antisense HNF-4 constructs decreased the apoCII promoter activity to 25–40% of the control, indicating that HNF-4 is a positive regulator of the apoCII gene. ARP-1 repressed the −205/+18 but not the −104/+18 apoCII promoter activity in HepG2 cells, indicating that the repression depends on the regulatory element CIIC. In contrast, combination of ARP-1 and HNF-4 transactivated different apoCII promoter segments as well as a minimal adenovirus major late promoter driven by the regulatory element CIIB. Mutagenesis or deletion of elements CIIB or CIIC established that the observed transactivation requires DNA binding of one of the two factors and may result from HNF-4-ARP-1 interactions that elicit the transactivation functions of HNF-4. The combined data indicate that RXRα/T3Rβ in the presence of T3 and HNF-4 can upregulate the apoCII promoter activity by binding to the regulatory elements CIIC and CIIB, respectively. In addition, ARP-1 can either have inhibitory or stimulatory effects on the apoCII promoter activity via different mechanisms.

Pleiotropic Functions of HDL Lead to Protection from Atherosclerosis and Other Diseases

High density lipoprotein (HDL) is a macromolecular complex of proteins and lipids that is produced primarily by the liver through a complex pathway that requires initially the functions of apolipoprotein A-I (apoA-I), ATP binding cassette transporter A1 (ABCA1) and lecithin:cholesterol acetyl transferase (LCAT) (Zannis et al., 2006b). Following synthesis, HDL affects the functions of the arterial wall cells through signaling mechanisms mediated by scavenger receptor class B type-I (SR-BI) and other cell surface proteins. The impetus for studying HDL has been the inverse correlation that exists between plasma HDL levels and the risk for coronary artery disease (CAD) (Gordon et al., 1989). HDL promotes cholesterol efflux (Gu et al., 2000; Nakamura et al., 2004), prevents oxidation of low density lipoprotein (LDL) (Navab et al., 2000a; Navab et al., 2000b), inhibits expression of proinflammatory cytokines by macrophages (Okura et al., 2010) as well as expression of adhesion molecules by endothelial cells (Cockerill et al., 1995; Nicholls et al,. 2005b). HDL inhibits cell apoptosis (Nofer et al., 2001) and promotes endothelial cell proliferation and migration (Seetharam et al., 2006). HDL stimulates release of nitric oxide (NO) from endothelial cells thus promoting vasodilation (Mineo et al., 2003). HDL also inhibits platelet aggregation and thrombosis (Dole et al., 2008) and has antibacterial, antiparasitic and antiviral activities (Parker et al., 1995; Singh et al., 1999; Vanhollebeke and Pays, 2010). Due to these properties HDL is thought to protect the endothelium and inhibit several steps in the cascade of events that lead to the pathogenesis of atherosclerosis and various other human diseases. This review focuses on two important aspects of contemporary HDL research. The first part considers briefly the structure of apoA-I and HDL and the key proteins that participate in the pathway of the biogenesis of HDL as well as clinical phenotypes associated with HDL abnormalities. The second part considers various physiological functions of HDL and apoA-I and the protective role of HDL against atherosclerosis and other diseases.