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Hepatocyte nuclear factor-4alpha (HNF4alpha) exists in multiple isoforms that are generated by alternative promoter (P1 and P2) usage and splicing. Here we establish monoclonal antibodies (mAbs) for detecting P1 and P2 promoter-driven HNF4alpha, and evaluate their expression in normal adult human tissues and surgically resected carcinomas of different origins. Using immunohistochemical analysis, we demonstrate that, while P1 promoter-driven HNF4alpha is expressed in hepatocytes, small intestine, colon, kidney and epididymis, P2 promoter-driven HNF4alpha is expressed in bile duct, pancreas, stomach, small intestine, colon and epididymis. Altered expression patterns of P1 and P2 promoter-driven HNF4alpha were observed in gastric, hepatocellular and colorectal carcinomas. HNF4alpha was expressed in lung metastases from renal cell, hepatocellular and colorectal carcinoma but was not observed in lung tumours. The P1 and P2 promoter-driven HNF4alpha expression pattern of tumour metastases correlated with the primary site of origin. P1 promoter-driven HNF4alpha was also found in intestinal metaplasia of the stomach. These data provide evidence for the tissue distribution of P1 and P2 promoter-driven HNF4alpha at the protein level and suggest that HNF4alpha may be a novel diagnostic marker for metastases of unknown primary. We propose that the dysregulation of alternative promoter usage of HNF4alpha is associated with the pathogenesis of certain cancers.

The physiological roles of the factor H (FH)-related proteins are controversial and poorly understood. Based on genetic studies, FH-related protein 5 (CFHR5) is implicated in glomerular diseases, such as atypical hemolytic uremic syndrome, dense deposit disease, and CFHR5 nephropathy. CFHR5 was also identified in glomerular immune deposits at the protein level. For CFHR5, weak complement regulatory activity and competition for C3b binding with the plasma complement inhibitor FH have been reported, but its function remains elusive. In this study, we identify pentraxin 3 (PTX3) as a novel ligand of CFHR5. Binding of native CFHR5 to PTX3 was detected in human plasma and the interaction was characterized using recombinant proteins. The binding of PTX3 to CFHR5 is of ∼2-fold higher affinity compared with that of FH. CFHR5 dose-dependently inhibited FH binding to PTX3 and also to the monomeric, denatured form of the short pentraxin C–reactive protein. Binding of PTX3 to CFHR5 resulted in increased C1q binding. Additionally, CFHR5 bound to extracellular matrix in vitro in a dose-dependent manner and competed with FH for binding. Altogether, CFHR5 reduced FH binding and its cofactor activity on pentraxins and the extracellular matrix, while at the same time allowed for enhanced C1q binding. Furthermore, CFHR5 allowed formation of the alternative pathway C3 convertase and supported complement activation. Thus, CFHR5 may locally enhance complement activation via interference with the complement-inhibiting function of FH, by enhancement of C1q binding, and by activating complement, thereby contributing to glomerular disease.

Cholesterol homeostasis is maintained by coordinate regulation of cholesterol synthesis and its conversion to bile acids in the liver. The excretion of cholesterol from liver and intestine is regulated by ATP-binding cassette half-transporters ABCG5 and ABCG8. The genes for these two proteins are closely linked and divergently transcribed from a common intergenic promoter region. Here, we identified a binding site for hepatocyte nuclear factor 4␣ (HNF4␣) in the ABCG5/ABCG8 intergenic promoter, through which HNF4␣ strongly activated the expression of a reporter gene in both directions. The HNF4␣-responsive element is flanked by two conserved GATA boxes that were also required for stimulation by HNF4␣. GATA4 and GATA6 bind to the GATA boxes, coexpression of GATA4 and HNF4␣ leads to a striking synergistic activation of both the ABCG5 and the ABCG8 promoters, and binding sites for HNF4␣ and GATA were essential for maximal synergism. We also show that HNF4␣, GATA4, and GATA6 colocalize in the nuclei of HepG2 cells and that a physical interaction between HNF4␣ and GATA4 is critical for the synergistic response. This is the first demonstration that HNF4␣ acts synergistically with GATA factors to activate gene expression in a bidirectional fashion.Cholesterol homeostasis is maintained by a series of regulatory pathways that control the synthesis of endogenous cholesterol, the absorption of dietary sterol, and the elimination of cholesterol and its catabolic end products, bile acids. Transcriptional control of many genes vital to these processes can be attributed to two classes of transcription factors: sterol regulatory element-binding proteins (SREBPs), especially SREBP-2, which control the production of key enzymes in cholesterol biosynthesis (11,36,38,39), and the nuclear hormone receptor family, including liver X receptor (LXR), farnesoid X receptor, small heterodimer partner, liver receptor homolog1 (LRH-1), and hepatocyte nuclear factor 4␣ (HNF4␣), which control the expression of genes involved in cholesterol efflux, catabolism, and elimination (3, 27).HNF4␣ is the most abundant nuclear orphan receptor expressed in the liver, and it is involved in early liver development (22). HNF4␣ is also expressed in kidney, intestine, and pancreas and is required for expression of many tissue-specific traits in all of these organs. Transcriptional activation by HNF4␣ is mediated by its binding as a homodimer to a DNA sequence composed of two direct repeats (DRs) of the hexanucleotide motif AGGTCA separated by 1 base, referred to as an HNF4␣ response element of the DR-1 type. Like other nuclear receptors, HNF4␣ exhibits a modular structure with six distinct domains (A to F). The N-terminal A/B domain is highly variable among nuclear receptors and contains a ligandindependent activation function 1 (AF-1) domain. The highly conserved C domain encodes the DNA binding domain of nuclear receptors and confers sequence-specific DNA recognition. By linking the highly structured C and E domains, the hinge D region may allow for flexibil...

Humoral fluid phase pattern recognition molecules (PRMs) are a key component of the activation and regulation of innate immunity. Humoral PRMs are diverse. We focused on the long pentraxin PTX3 as a paradigmatic example of fluid phase PRMs. PTX3 acts as a functional ancestor of antibodies and plays a non-redundant role in resistance against selected microbes in mouse and man and in the regulation of inflammation. This molecule interacts with complement components, thus modulating complement activation. In particular PTX3 regulates complement-driven macrophage-mediated tumor progression, acting as an extrinsic oncosuppressor in preclinical models and selected human tumors. Evidence collected over the years suggests that PTX3 is a biomarker and potential therapeutic agent in humans, and pave the way to translation of this molecule into the clinic.

Pentraxins are a family of multimeric proteins characterized by the presence of a pentraxin signature in their C-terminus region. Based on the primary structure, pentraxins are divided into short and long pentraxin: C-reactive protein (CRP) is the prototype of the short pentraxin subfamily while pentraxin 3 (PTX3) is the prototypic long pentraxin. Despite these two molecules exert similar fundamental actions in the regulation of innate immune and inflammatory responses, several differences exist between CRP and PTX3, including gene organization, protein oligomerization and expression pattern. The pathophysiological roles of PTX3 have been investigated using genetically modified mice since PTX3 gene organization and regulation are well conserved between mouse and human. Such in vivo studies figured out that PTX3 mainly have host-protective effects, even if it could also exert negative effects under certain pathophysiologic conditions. Here we will review the general properties of CRP and PTX3, emphasizing the differences between the two molecules and the regulatory functions exerted by PTX3 in innate immunity and inflammation.

Pentraxin 3 (PTX3), a long pentraxin subfamily member in the pentraxin family, plays an important role in innate immunity as a soluble pattern recognition receptor. Plasma PTX3 is elevated in sepsis (∼200 ng/ml) and correlates with mortality. The roles of PTX3 in sepsis, however, are not well understood. To investigate the ligands of PTX3 in sepsis, we performed a targeted proteomic study of circulating PTX3 complexes using magnetic bead-based immunopurification and shotgun proteomics for label-free relative quantitation via spectral counting. From septic patient fluids, we successfully identified 104 candidate proteins, including the known PTX3-interacting proteins involved in complement activation, pathogen opsonization, inflammation regulation, and extracellular matrix deposition. Notably, the proteomic profile additionally showed that PTX3 formed a complex with some of the components of neutrophil extracellular traps. Subsequent biochemical analyses revealed a direct interaction of bactericidal proteins azurocidin 1 (AZU1) and myeloperoxidase with PTX3. AZU1 exhibited high affinity binding (KD = 22 ± 7.6 nm) to full-length PTX3 in a calcium ion-dependent manner and bound specifically to an oligomer of the PTX3 N-terminal domain. Immunohistochemistry with a specific monoclonal antibody generated against AZU1 revealed a partial co-localization of AZU1 with PTX3 in neutrophil extracellular traps. The association of circulating PTX3 with components of the neutrophil extracellular traps in sepsis suggests a role for PTX3 in host defense and as a potential diagnostic target.

Hepatocyte nuclear factor-4α (HNF4α, NR2A1) is a nuclear receptor that has a critical role in hepatocyte differentiation and the maintenance of homeostasis in the adult liver. However, a detailed understanding of native HNF4α in the steady-state remains to be elucidated. Here we report the native HNF4α isoform, phosphorylation status, and complexes in the steady-state, as shown by shotgun proteomics in HepG2 hepatocarcinoma cells. Shotgun proteomic analysis revealed the complexity of native HNF4α, including multiple phosphorylation sites and inter-isoform heterodimerization. The associating complexes identified by label-free semiquantitative proteomic analysis include the following: the DNA-dependent protein kinase catalytic subunit, histone acetyltransferase complexes, mRNA splicing complex, other nuclear receptor coactivator complexes, the chromatin remodeling complex, and the nucleosome remodeling and histone deacetylation complex. Among the associating proteins, GRB10 interacting GYF protein 2 (GIGYF2, PERQ2) is a new candidate cofactor in metabolic regulation. Moreover, an unexpected heterodimerization of HNF4α and hepatocyte nuclear factor-4γ was found. A biochemical and genomewide analysis of transcriptional regulation showed that this heterodimerization activates gene transcription. The genes thus transcribed include the cell death-inducing DEF45-like effector b (CIDEB) gene, which is an important regulator of lipid metabolism in the liver. This suggests that the analysis of the distinctive stoichiometric balance of native HNF4α and its cofactor complexes described here are important for an accurate understanding of transcriptional regulation.