Search

Inhalation of traffic-related particulate matter (e.g., diesel exhaust particles [DEPs]) is associated with acute inflammatory responses in the lung, and it promotes the development and aggravation of allergic airway diseases. We previously demonstrated that exposure to DEP was associated with increased recruitment and maturation of monocytes and conventional dendritic cells (DCs), resulting in TH2 polarization. Monocytes and immature DCs express the G-protein coupled receptor chemR23, which binds the chemoattractant chemerin. Using chemR23 knockout (KO) and corresponding wild-type (WT) mice, we determined the role of chemR23 signaling in response to acute exposure to DEPs and in response to DEP-enhanced house dust mite (HDM)-induced allergic airway inflammation. Exposure to DEP alone, as well as combined exposure to DEP plus HDM, elevated the levels of chemerin in the bronchoalveolar lavage fluid of WT mice. In response to acute exposure to DEPs, monocytes and monocyte-derived DCs accumulated in the lungs of WT mice, but this response was significantly attenuated in chemR23 KO mice. Concomitant exposure to DEP plus HDM resulted in allergic airway inflammation with increased eosinophilia, goblet cell metaplasia, and TH2 cytokine production in WT mice, which was further enhanced in chemR23 KO mice. In conclusion, we demonstrated an opposing role for chemR23 signaling depending on the context of DEP-induced inflammation. The chemR23 axis showed proinflammatory properties in a model of DEP-induced acute lung inflammation, in contrast to anti-inflammatory effects in a model of DEP-enhanced allergic airway inflammation., (Copyright © 2016 by The American Association of Immunologists, Inc.)

Aims/hypothesis: Glucokinase plays a key role in glucose homeostasis and the expression of its gene is differentially regulated in pancreatic beta cells and in the liver through distinct promoters. The factors that determine the tissue-specific expression of the glucokinase gene are not known. Putative binding sites for hepatocyte nuclear factor (HNF)-6, the prototype of the ONECUT family of transcription factors, are present in the hepatic promoter of the glucokinase gene and hnf6 knockout mice are diabetic [corrected]. We hypothesized that HNF-6 controls the activity of the hepatic glucokinase promoter., Methods: We tested the binding of recombinant HNF-6 to DNA sequences from the mouse hepatic glucokinase promoter in vitro and the effect of HNF-6 on promoter activity in transfected cells. We investigated in vivo the role of HNF-6 in mice by examining the effect of inactivating the hnf6 gene on glucokinase gene-specific deoxyribonuclease I hypersensitive sites in liver chromatin and on liver glucokinase mRNA concentration., Results: HNF-6 bound to the hepatic promoter of the glucokinase gene and stimulated its activity. Inactivation of the hnf6 gene did not modify the pattern of deoxyribonuclease I hypersensitive sites but was associated with a decrease of liver glucokinase mRNA to half the control value., Conclusions/interpretation: Although HNF-6 is not required to open chromatin of the hepatic promoter of the glucokinase gene, it stimulates transcription of the glucokinase gene in the liver. This could partly explain the diabetes observed in hnf6 knockout mice.

During liver development, hepatoblasts differentiate into hepatocytes or biliary epithelial cells (BEC). The BEC delineate the intrahepatic and extrahepatic bile ducts, and the gallbladder. The transcription factors that control the development of the biliary tract are unknown. Previous work has shown that the onecut transcription factor HNF6 is expressed in hepatoblasts and in the gallbladder primordium. We now show that HNF6 is also expressed in the BEC of the developing intrahepatic bile ducts, and investigate its involvement in biliary tract development by analyzing the phenotype of Hnf6(-/-) mice. In these mice, the gallbladder was absent, the extrahepatic bile ducts were abnormal and the development of the intrahepatic bile ducts was perturbed in the prenatal period. The morphology of the intrahepatic bile ducts was identical to that seen in mice whose Hnf1beta gene has been conditionally inactivated in the liver. HNF1beta expression was downregulated in the intrahepatic bile ducts of Hnf6(-/-) mice during development. Furthermore, we found that HNF6 can stimulate the Hnf1beta promoter. We conclude that HNF6 is essential for differentiation and morphogenesis of the biliary tract and that intrahepatic bile duct development is controlled by a HNF6-->HNF1beta cascade.

Microphthalmia-associated transcription factor (MITF) is essential for melanocyte differentiation. MITF mutations are associated with some cases of Waardenburg syndrome (WS) type 2. WS is a dominantly inherited disease characterized by auditory-pigmentary defects that result from the absence of melanocytes. The lack of mutation in MITF coding sequences in some WS2 patients suggests that unidentified factors controlling MITF expression might be involved. We show here that the cut-homeodomain transcription factor Onecut-2 (OC-2) is expressed in melanocytes and binds to the MITF gene promoter. Overexpression of OC-2 in transfected cells stimulates MITF promoter activity. Mutations that prevent OC-2 binding decrease MITF promoter activity by 75%. Based on these results, we searched in 56 WS2 patients for mutations in the OC2 gene or in OC-2 binding sites in the MITF promoter, but none was found. These results show that OC-2 stimulates MITF expression and that OC2 is a candidate gene, but not a common cause, of WS., (Copyright 2001 Academic Press.)

Transcription factors of the ONECUT class, whose prototype is HNF-6, contain a single cut domain and a divergent homeodomain characterized by a phenylalanine at position 48 and a methionine at position 50. The cut domain is required for DNA binding. The homeodomain is required either for DNA binding or for transcriptional stimulation, depending on the target gene. Transcriptional stimulation by the homeodomain involves the F48M50 dyad. We investigate here how HNF-6 stimulates transcription. We identify transcriptionally active domains of HNF-6 that are conserved among members of the ONECUT class and show that the cut domain of HNF-6 participates to DNA binding and, via a LXXLL motif, to transcriptional stimulation. We also demonstrate that, on a target gene to which HNF-6 binds without requirement for the homeodomain, transcriptional stimulation involves an interaction of HNF-6 with the coactivator CREB-binding protein (CBP). This interaction depends both on the LXXLL motif of the cut domain and on the F48M50 dyad of the homeodomain. On a target gene for which the homeodomain is required for DNA binding, but not for transcriptional stimulation, HNF-6 interacts with the coactivator p300/CBP-associated factor but not with CBP. These data show that a transcription factor can act via different, sequence-specific, mechanisms that combine distinct modes of DNA binding with the use of different coactivators.

Transcription factors of the ONECUT class, whose prototype is hepatocyte nuclear factor (HNF)-6, are characterized by the presence of a single cut domain and by a peculiar homeodomain (Lannoy, V. J., Bürglin, T. R., Rousseau, G. G., and Lemaigre, F. P. (1998) J. Biol. Chem. 273, 13552-13562). We report here the identification and characterization of human OC-2, the second mammalian member of this class. The OC-2 gene is located on human chromosome 18. The distribution of OC-2 mRNA in humans is tissue-restricted, the strongest expression being detected in the liver and skin. The amino acid sequence of OC-2 contains several regions of high similarity to HNF-6. The recognition properties of OC-2 for binding sites present in regulatory regions of liver-expressed genes differ from, but overlap with, those of HNF-6. Like HNF-6, OC-2 stimulates transcription of the hnf-3beta gene in transient transfection experiments, suggesting that OC-2 participates in the network of transcription factors required for liver differentiation and metabolism.

Hepatocyte nuclear factor-6 (HNF-6) contains a single cut domain and a homeodomain characterized by a phenylalanine at position 48 and a methionine at position 50. We describe here two isoforms of HNF-6 which differ by the linker that separates these domains. Both isoforms stimulated transcription. The affinity of HNF-6alpha and HNF-6beta for DNA differed, depending on the target sequence. Binding of HNF-6 to DNA involved the cut domain and the homeodomain, but the latter was not required for binding to a subset of sites. Mutations of the F48M50 dyad that did not affect DNA binding reduced the transcriptional stimulation of constructs that do not require the homeodomain for DNA binding, but did not affect the stimulation of constructs that do require the homeodomain. Comparative trees of mammalian, Drosophila, and Caenorhabditis elegans proteins showed that HNF-6 defines a new class, which we call ONECUT, of homeodomain proteins. C. elegans proteins of this class bound to HNF-6 DNA targets. Thus, depending on their sequence, these targets determine for HNF-6 at least two modes of DNA binding, which hinge on the homeodomain and on the linker that separates it from the cut domain, and two modes of transcriptional stimulation, which hinge on the homeodomain.

Protein C is a vitamin K-dependent zymogen of a serine protease that inhibits blood coagulation by proteolytic inactivation of factors Va and VIIIa. Individuals affected by protein C deficiency are at risk for venous thrombosis. One such affected individual was shown earlier to carry a -14 T --> C mutation in the promoter region of the protein C gene. It is shown here that the region around this mutation corresponds to a binding site for the transcription factor hepatocyte nuclear factor (HNF)-6 and that this site completely overlaps an HNF-1 binding site. HNF-6 and HNF-1 bound in a mutually exclusive manner. The -14 T --> C mutation reduced HNF-6 binding. In transient transfection experiments, HNF-6 transactivated the wild-type protein C promoter and introduction of the mutation abolished transactivation by HNF-6. Similar experiments showed that wild-type protein C promoter activity was reduced by cotransfection of an HNF-1 expression vector. This inhibiting effect of HNF-1 was reversed to a stimulatory effect when promoter sequences either upstream or downstream of the HNF-6/HNF-1 site were deleted. It is concluded that HNF-6 is a major determinant of protein C gene activity. Moreover, this is the first report describing the putative involvement of HNF-6 and of an HNF-6 binding site in human pathology.

Tissue-specific transcription is regulated in part by cell type-restricted proteins that bind to defined sequences in target genes. The DNA-binding domain of these proteins is often evolutionarily conserved. On this basis, liver-enriched transcription factors were classified into five families. We describe here the mammalian prototype of a sixth family, which we therefore call hepatocyte nuclear factor 6 (HNF-6). It activates the promoter of a gene involved in the control of glucose metabolism. HNF-6 contains two different DNA-binding domains. One of these corresponds to a novel type of homeodomain. The other is homologous to the Drosophila cut domain. A similar bipartite sequence is coded by the genome of Caenorhabditis elegans.