Inflammation-mediated deacetylation of the ribonuclease 1 promoter via histone deacetylase 2 in endothelial cells.

Ribonuclease 1 (RNase1) is a circulating extracellular endonuclease that regulates the vascular homeostasis of extracellular RNA and acts as a vessel- and tissue-protective enzyme. Upon long-term inflammation, high amounts of proinflammatory cytokines affect endothelial cell (EC) function by down-regulation of RNase1. Here, we investigated the transcriptional regulation of RNase1 upon inflammation in HUVECs. TNF-α or IL-1ß stimulation reduced the expression of RNase1 relative to the acetylation state of histone 3 at lysine 27 and histone 4 of the RNASE1 promoter. Inhibition of histone deacetylase (HDAC) 1, 2, and 3 by the specific class I HDAC inhibitor MS275 abolished the TNF-α- or IL-1ß-mediated effect on the mRNA and chromatin levels of RNase1. Moreover, chromatin immunoprecipitation kinetics revealed that HDAC2 accumulates at the RNASE1 promoter upon TNF-α stimulation, indicating an essential role for HDAC2 in regulating RNase1 expression. Thus, proinflammatory stimulation induced recruitment of HDAC2 to attenuate histone acetylation at the RNASE1 promoter site. Consequently, treatment with HDAC inhibitors may provide a new therapeutic strategy to stabilize vascular homeostasis in the context of inflammation by preventing RNase1 down-regulation in ECs.-Bedenbender, K., Scheller, N., Fischer, S., Leiting, S., Preissner, K. T., Schmeck, B. T., Vollmeister, E. Inflammation-mediated deacetylation of the ribonuclease 1 promoter via histone deacetylase 2 in endothelial cells.

Transforming Growth Factor-ß (TGF-ß) Inhibits the Expression of Factor VII-activating Protease (FSAP) in Hepatocytes.

Deletion of the Habp2 gene encoding Factor VII-activating protease (FSAP) increases liver fibrosis in mice. A single nucleotide polymorphism (G534E) in HABP2 leads to lower enzymatic activity and is associated with enhanced liver fibrosis in humans. Liver fibrosis is associated with a decrease in FSAP expression but, to date, nothing is known about how this might be regulated. Primary mouse hepatocytes or the hepatocyte cell line, AML12, were treated with different factors, and expression of FSAP was determined. Of the various regulatory factors tested, only transforming growth factor-ß (TGF-ß) demonstrated a concentration- and time-dependent inhibition of FSAP expression at the mRNA and protein level. The TGF-ß-Type I receptor (ALK-5) antagonist SB431542 and Smad2 siRNA, but neither SIS3, which inhibits SMAD3, nor siRNA against Smad3 could block this effect. Various regions of the HABP2 promoter region were cloned into reporter constructs, and the promoter activity was determined. Accordingly, the promoter activity, which could phenocopy changes in Habp2 mRNA in response to TGF-ß, was found to be located in the 177-bp region upstream of the transcription start site, and this region did not contain any SMAD binding sites. Mutation analysis of the promoter and chromatin immunoprecipitation assays were performed to identify an important role for the ATF3 binding element. Thus, TGF-ß is the most likely mediator responsible for the decrease in FSAP expression in liver fibrosis.

Role of the Steroid Sulfate Uptake Transporter Soat (Slc10a6) in Adipose Tissue and 3T3-L1 Adipocytes.

In addition to the endocrine and paracrine systems, peripheral tissues such as gonads, skin, and adipose tissue are involved in the intracrine mechanisms responsible for the formation of sex steroids via the transformation of dehydroepiandrosterone and dehydroepiandrosterone sulfate (DHEA/DHEAS) into potent androgenic and estrogenic hormones. Numerous studies have examined the relationship between overweight, central obesity, and plasma levels of DHEA and DHEAS. The sodium-dependent organic anion transporter Soat (Slc10a6) is a plasma membrane uptake transporter for sulfated steroids. Significantly increased expression of Slc10a6 mRNA has been previously described in organs and tissues of lipopolysaccharide (LPS)-treated mice, including white adipose tissue. These findings suggest that Soat plays a role in the supply of steroids in peripheral target tissues. The present study aimed to investigate the expression of Soat in adipocytes and its role in adipogenesis. Soat expression was analyzed in mouse white intra-abdominal (WAT), subcutaneous (SAT), and brown (BAT) adipose tissue samples and in murine 3T3-L1 adipocytes. In addition, adipose tissue mass and size of the adipocytes were analyzed in wild-type and Slc10a6 -/- knockout mice. Soat expression was detected in mouse WAT, SAT, and BAT using immunofluorescence. The expression of Slc10a6 mRNA was significantly higher in 3T3-L1 adipocytes than that of preadipocytes and was significantly upregulated by exposure to lipopolysaccharide (LPS). Slc10a6 mRNA levels were also upregulated in the adipose tissue of LPS-treated mice. In Slc10a6 -/- knockout mice, adipocytes increased in size in the WAT and SAT of female mice and in the BAT of male mice, suggesting adipocyte hypertrophy. The serum levels of adiponectin, resistin, and leptin were comparable in wild-type and Slc10a6 -/- knockout mice. The treatment of 3T3-L1 adipocytes with DHEA significantly reduced lipid accumulation, while DHEAS did not have a significant effect. However, following LPS-induced Soat upregulation, DHEAS also significantly inhibited lipid accumulation in adipocytes. In conclusion, Soat-mediated import of DHEAS and other sulfated steroids could contribute to the complex pathways of sex steroid intracrinology in adipose tissues. Although in cell cultures the Soat-mediated uptake of DHEAS appears to reduce lipid accumulation, in Slc10a6 -/- knockout mice, the Soat deletion induced adipocyte hyperplasia through hitherto unknown mechanisms.

Tyrosine 146 of the Human Na+/Taurocholate Cotransporting Polypeptide (NTCP) Is Essential for Its Hepatitis B Virus (HBV) Receptor Function and HBV Entry into Hepatocytes.

Na+/taurocholate cotransporting polypeptide (NTCP, gene symbol SLC10A1) is a hepatic bile acid uptake carrier participating in the enterohepatic circulation of bile acids. Apart from its transporter function, NTCP acts as the high-affinity liver-specific receptor for the hepatitis B virus (HBV), which attaches via its preS1-peptide domain of the large surface protein to NTCP, subsequently leading to endocytosis of the virus/NTCP-receptor complex. Although the process of NTCP-dependent HBV infection of hepatocytes has received much attention over the last decade, the precise molecular sites of the virus/NTCP interaction have not been fully identified. Inspection of the primary protein sequence of human NTCP revealed 139YIYSRGIY146 as a highly conserved tyrosine-rich motif. To study the role of Y139, Y141 and Y146 amino acids in NTCP biology, the aforementioned residues were substituted with alanine, phenylalanine or glutamate (mimicking phosphorylation) using site-directed mutagenesis. Similar to wt NTCP, the Y139A, Y141A, Y146A, Y141F, Y146F, and Y146E mutants were expressed at the plasma membrane of HEK293 cells and exhibited intact bile acid transport function. Y146A, Y146E, and Y146F demonstrated transport kinetics comparable to wild-type NTCP with Km values of 57.3-112.4 µM and Vmax values of 6683-7579 pmol/mg protein/min. Only Y141E was transport deficient, most likely due to an intracellular accumulation of the mutant protein. Most importantly, Y146A and Y146E mutation completely abrogated binding of the viral preS1-peptide to NTCP, while the Y146F mutant of NTCP showed some residual binding competence for preS1. Consequently, the NTCP mutants Y146A and Y146E, when expressed in HepG2 hepatoma cells, showed complete loss of susceptibility for in vitro HBV infection. In conclusion, tyrosine 146, and to some extent tyrosine 141, both belonging to the tyrosine-rich motif 139YIYSRGIY146 of human NTCP, are newly identified amino acid residues that play an essential role in the interaction of HBV with its receptor NTCP and, thus, in the process of virus entry into hepatocytes.

IFITM3 Interacts with the HBV/HDV Receptor NTCP and Modulates Virus Entry and Infection.

The Na+/taurocholate co-transporting polypeptide (NTCP, gene symbol SLC10A1) is both a physiological bile acid transporter and the high-affinity hepatic receptor for the hepatitis B and D viruses (HBV/HDV). Virus entry via endocytosis of the virus/NTCP complex involves co-factors, but this process is not fully understood. As part of the innate immunity, interferon-induced transmembrane proteins (IFITM) 1-3 have been characterized as virus entry-restricting factors for many viruses. The present study identified IFITM3 as a novel protein-protein interaction (PPI) partner of NTCP based on membrane yeast-two hybrid and co-immunoprecipitation experiments. Surprisingly, IFITM3 knockdown significantly reduced in vitro HBV infection rates of NTCP-expressing HuH7 cells and primary human hepatocytes (PHHs). In addition, HuH7-NTCP cells showed significantly lower HDV infection rates, whereas infection with influenza A virus was increased. HBV-derived myr-preS1 peptide binding to HuH7-NTCP cells was intact even under IFITM3 knockdown, suggesting that IFITM3-mediated HBV/HDV infection enhancement occurs in a step subsequent to the viral attachment to NTCP. In conclusion, IFITM3 was identified as a novel NTCP co-factor that significantly affects in vitro infection with HBV and HDV in NTCP-expressing hepatoma cells and PHHs. While there is clear evidence for a direct PPI between IFITM3 and NTCP, the specific mechanism by which this PPI facilitates the infection process remains to be identified in future studies.

Sequencing of the Canine Cytochrome P450 CYP2C41 Gene and Genotyping of Its Polymorphic Occurrence in 36 Dog Breeds.

Cytochrome P450 (CYP) drug metabolizing enzymes play an important role in efficient drug metabolism and elimination. Many CYPs are polymorphic and, thereby, drug metabolism can vary between individuals. In the case of canine CYP2C41, gene polymorphism was identified. However, as the first available canine genome sequences all were CYP2C41 negative, this polymorphism could not be clarified at the genomic level. The present study provides an exact characterization of the CYP2C41 gene deletion polymorphism at the genomic level and presents a PCR-based genotyping method that was used for CYP2C41 genotyping of 1,089 individual subjects from 36 different dog breeds. None of the Bearded Collie, Bernese Mountain, Boxer, Briard, French Bulldog or Irish Wolfhound subjects had the CYP2C41 gene in their genomes. In contrast, in the Chinese Char-Pei, Siberian Husky, Schapendoes and Kangal breeds, the CYP2C41 allele frequency was very high, with values of 67, 57, 43, and 34%, respectively. Interestingly, the site of gene deletion was identical for all CYP2C41 negative dogs, and all CYP2C41 positive dogs showed highly homologous sequence domains upstream and downstream from the CYP2C41 gene. CYP2C41 genotyping can now be routinely used in future pharmacokinetic studies in canines, in order to identify genetically-based poor or extensive drug metabolizers. This, together with more extensive in vitro drug screening for CYP2C41 substrates will help to determine the clinical relevance of CYP2C41, and to optimize drug treatment. Although the relative abundance of the CYP2C41 protein in the canine liver seems to not be very high, this CYP could substantially contribute to hepatic drug metabolism in dogs expressing CYP2C41 from both alleles and, when CYP2C41 shows higher catalytic activity to a given drug than other hepatic metabolic enzymes.

Detection of the ABCB11930_1931del TC Mutation in Two Suspected Ivermectin-Sensitive Cats and Their Relatives by a Novel TaqMan Allelic Discrimination Assay.

The multidrug resistance gene MDR1 (syn. ABCB1) encodes for the multidrug efflux transporter P-glycoprotein (P-gp), which is highly expressed at the blood-brain barrier and protects the brain from potentially neurotoxic compounds, such as ivermectin. MDR1 mutation in dogs is known to be linked to dramatically increased brain accumulation of ivermectin and life-threatening neurological toxicity. The present report describes two suspected ivermectin-sensitive Maine Coon cats, which exhibited neurological toxicity following subcutaneous application of therapeutic doses of ivermectin. Both cats showed a homozygous 2-bp deletion in the MDR1/ABCB1 coding sequence (ABCB11930_1931del TC, syn. MDR1 nt1930(del2)) that had previously been associated with a drug-sensitive phenotype in cats. For cat MDR1 genotyping, a novel TaqMan allelic discrimination assay was established and validated. This assay was used for ABCB11930_1931del TC genotyping of the drug-sensitive cats as well as of more than 50 relatives. About half of them had the heterozygous MDR1(+/-) genotype, while none of these related cats with former ivermectin treatment had a history of drug-sensitivity. In conclusion: The present study supports previous findings on drug-sensitivity in cats with homozygous ABCB11930_1931del TC mutation. The newly established TaqMan allelic discrimination assay provides a useful and reliable method for routine MDR1 genotyping in cats in order to identify drug-sensitive cats prior to treatment with established P-gp substrates such as ivermectin and other macrocyclic lactones and thus to improve therapeutic safety.

The orphan solute carrier SLC10A7 is a novel negative regulator of intracellular calcium signaling.

SLC10A7 represents an orphan member of the Solute Carrier Family SLC10. Recently, mutations in the human SLC10A7 gene were associated with skeletal dysplasia, amelogenesis imperfecta, and decreased bone mineral density. However, the exact molecular function of SLC10A7 and the mechanisms underlying these pathologies are still unknown. For this reason, the role of SLC10A7 on intracellular calcium signaling was investigated. SLC10A7 protein expression was negatively correlated with store-operated calcium entry (SOCE) via the plasma membrane. Whereas SLC10A7 knockout HAP1 cells showed significantly increased calcium influx after thapsigargin, ionomycin and ATP/carbachol treatment, SLC10A7 overexpression reduced this calcium influx. Intracellular Ca2+ levels were higher in the SLC10A7 knockout cells and lower in the SLC10A7-overexpressing cells. The SLC10A7 protein co-localized with STIM1, Orai1, and SERCA2. Most of the previously described human SLC10A7 mutations had no effect on the calcium influx and thus were confirmed to be functionally inactive. In the present study, SLC10A7 was established as a novel negative regulator of intracellular calcium signaling that most likely acts via STIM1, Orai1 and/or SERCA2 inhibition. Based on this, SLC10A7 is suggested to be named as negative regulator of intracellular calcium signaling (in short: RCAS).

Self-extracellular RNA acts in synergy with exogenous danger signals to promote inflammation.

Self-extracellular RNA (eRNA), released from stressed or injured cells upon various pathological situations such as ischemia-reperfusion-injury, has been shown to act as an alarmin by inducing procoagulatory and proinflammatory responses. In particular, M1-polarization of macrophages by eRNA resulted in the expression and release of a variety of cytokines, including tumor necrosis factor (TNF)-α or interleukin-6 (IL-6). The present study now investigates in which way self-eRNA may influence the response of macrophages towards various Toll-like receptor (TLR)-agonists. Isolated agonists of TLR2 (Pam2CSK4), TLR3 (PolyIC), TLR4 (LPS), or TLR7 (R848) induced the release of TNF-α in a concentration-dependent manner in murine macrophages, differentiated from bone marrow-derived stem cells by mouse colony stimulating factor. Here, the presence of eRNA shifted the dose-response curve for Pam2CSK4 (Pam) considerably to the left, indicating that eRNA synergistically enhanced the cytokine liberation from macrophages even at very low Pam-levels. The synergistic activation of TLR2 by eRNA/Pam was duplicated by other TLR2-agonists such as FSL-1 or Pam3CSK4. In contrast, for TLR4-agonists such as LPS a synergistic effect of eRNA was much weaker, and was not existent for TLR3-, or TLR7-agonists. The synergistic eRNA/Pam action was dependent on the NFκB-signaling pathway as well as on p38MAP- and MEK1/ERK-kinases and was prevented by predigestion of eRNA with RNase1 or by antibodies against TLR2. Thus, the presence of self-eRNA as alarming molecule sensitizes innate immune responses towards pathogen-associated molecular patterns (PAMPs) in a synergistic way and may thereby contribute to the differentiated outcome of inflammatory responses.

Structural requirements for the procoagulant activity of nucleic acids.

Nucleic acids, especially extracellular RNA, are exposed following tissue- or vessel damage and have previously been shown to activate the intrinsic blood coagulation pathway in vitro and in vivo. Yet, no information on structural requirements for the procoagulant activity of nucleic acids is available. A comparison of linear and hairpin-forming RNA- and DNA-oligomers revealed that all tested oligomers forming a stable hairpin structure were protected from degradation in human plasma. In contrast to linear nucleic acids, hairpin forming compounds demonstrated highest procoagulant activities based on the analysis of clotting time in human plasma and in a prekallikrein activation assay. Moreover, the procoagulant activities of the DNA-oligomers correlated well with their binding affinity to high molecular weight kininogen, whereas the binding affinity of all tested oligomers to prekallikrein was low. Furthermore, four DNA-aptamers directed against thrombin, activated protein C, vascular endothelial growth factor and nucleolin as well as the naturally occurring small nucleolar RNA U6snRNA were identified as effective cofactors for prekallikrein auto-activation. Together, we conclude that hairpin-forming nucleic acids are most effective in promoting procoagulant activities, largely mediated by their specific binding to kininogen. Thus, in vivo application of therapeutic nucleic acids like aptamers might have undesired prothrombotic or proinflammatory side effects.