The impact of the rs8005161 polymorphism on G protein-coupled receptor GPR65 (TDAG8) pH-associated activation in intestinal inflammation.

BACKGROUND: Tissue inflammation in inflammatory bowel diseases (IBD) is associated with a decrease in local pH. The gene encoding G-protein-coupled receptor 65 (GPR65) has recently been reported to be a genetic risk factor for IBD. In response to extracellular acidification, proton activation of GPR65 stimulates cAMP and Rho signalling pathways. We aimed to analyse the clinical and functional relevance of the GPR65 associated single nucleotide polymorphism (SNP) rs8005161. METHODS: 1138 individuals from a mixed cohort of IBD patients and healthy volunteers were genotyped for SNPs associated with GPR65 (rs8005161, rs3742704) and galactosylceramidase (rs1805078) by Taqman SNP assays. 2300 patients from the Swiss IBD Cohort Study (SIBDC) were genotyped for rs8005161 by mass spectrometry based SNP genotyping. IBD patients from the SIBDC carrying rs8005161 TT, CT, CC and non-IBD controls (CC) were recruited for functional studies. Human CD14+ cells were isolated from blood samples and subjected to an extracellular acidic pH shift, cAMP accumulation and RhoA activation were measured. RESULTS: In our mixed cohort, but not in SIBDC patients, the minor variant rs8005161 was significantly associated with UC. In SIBDC patients, we observed a consistent trend in increased disease severity in patients carrying the rs8005161-TT and rs8005161-CT alleles. No significant differences were observed in the pH associated activation of cAMP production between IBD (TT, CT, WT/CC) and non-IBD (WT/CC) genotype carriers upon an acidic extracellular pH shift. However, we observed significantly impaired RhoA activation after an extracellular acidic pH shift in IBD patients, irrespective of the rs8005161 allele. CONCLUSIONS: The T allele of rs8005161 might confer a more severe disease course in IBD patients. Human monocytes from IBD patients showed impaired pH associated RhoA activation upon an acidic pH shift.

The pH-sensing receptor OGR1 improves barrier function of epithelial cells and inhibits migration in an acidic environment.

The pH-sensing receptor ovarian cancer G protein-coupled receptor 1 (OGR1; GPR68) is expressed in the gut. Inflammatory bowel disease is typically associated with a decrease in local pH, which may lead to altered epithelial barrier function and subsequent gastrointestinal repair involving epithelial cell adhesion and migration. As the mechanisms underlying the response to pH changes are not well understood, we have investigated OGR1-mediated, pH-dependent signaling pathways in intestinal epithelial cells. Caco-2 cells stably overexpressing OGR1 were created and validated as tools to study OGR1 signaling. Barrier function, migration, and proliferation were measured using electric cell-substrate impedance-sensing technology. Localization of the tight junction proteins zonula occludens protein 1 and occludin and the rearrangement of cytoskeletal actin were examined by confocal microscopy. Paracellular permeability and protein and gene expression analysis using DNA microarrays were performed on filter-grown Caco-2 monolayers. We report that an acidic pH shift from pH 7.8 to 6.6 improved barrier function and stimulated reorganization of filamentous actin with prominent basal stress fiber formation. Cell migration and proliferation during in vitro wound healing were inhibited. Gene expression analysis revealed significant upregulation of genes related to cytoskeleton remodeling, cell adhesion, and growth factor signaling. We conclude that acidic extracellular pH can have a signaling function and impact the physiology of intestinal epithelial cells. The deconstruction of OGR1-dependent signaling may aid our understanding of mucosal inflammation mechanisms.

The organic solute transporters alpha and beta are induced by hypoxia in human hepatocytes.

BACKGROUND & AIMS: The organic solute transporters alpha and beta (OSTα-OSTß) form a heterodimeric transporter located at the basolateral membrane of intestinal epithelial cells and hepatocytes. Liver injury caused by ischaemia-reperfusion, cancer, inflammation or cholestasis can induce a state of hypoxia in hepatocytes. Here, we studied the effect of hypoxia on the expression of OSTα-OSTß. METHODS: OSTα-OSTß expression was measured in Huh7 cells and primary human hepatocytes (PHH) exposed to chenodeoxycholic acid (CDCA), hypoxia or both. OSTα-OSTß promoter activity was analysed in luciferase reporter gene assays. Binding of hypoxia-inducible factor-1 alpha (HIF-1α) to the OSTα-OSTß gene promoters was studied in electrophoretic mobility shift assays (EMSA). RESULTS: Expression of OSTα and OSTß increased in PHH under conditions of hypoxia. Exposure of Huh7 cells or PHH to CDCA (50 µM) enhanced the effect of hypoxia on OSTα mRNA levels. In luciferase assays and EMSA, the inducing effect of low oxygen could be assigned to HIF-1α, which binds to hypoxia responsive elements (HRE) in the OSTα and OSTß gene promoters. Site-directed mutagenesis of either the predicted HRE or the bile acid responsive FXR binding site abolished inducibility of the OSTα promoter, indicating that both elements need to be intact for induction by hypoxia and CDCA. In a rat model of chronic renal failure, the known increase in hepatic OSTα expression was associated with an increase in HIF-1α protein levels. CONCLUSION: OSTα-OSTß expression is induced by hypoxia. FXR and HIF-1α bind in close proximity to the OSTα gene promoter and produce synergistic effects on OSTα expression.

The SLCO1A2 gene, encoding human organic anion-transporting polypeptide 1A2, is transactivated by the vitamin D receptor.

Organic anion-transporting polypeptide 1A2 (OATP1A2) (gene symbol, SLCO1A2) mediates cellular uptake of a wide range of endogenous substrates, as well as drugs and xenobiotics. OATP1A2 is expressed in several tissues, including apical membranes of small intestinal epithelial cells. Given its role in intestinal drug absorption, a detailed analysis of the mechanisms that regulate SLCO1A2 gene expression is potentially of great pharmacological relevance. We show here that treatment of human intestine-derived Caco-2 cells with vitamin D(3) markedly increased endogenous OATP1A2 mRNA and protein levels. Suppression of endogenous vitamin D receptor (VDR) expression with siRNAs significantly reduced this induction. Two alternative promoter regions exist in genomic databases for the SLCO1A2 gene. One putative VDR response element (VDRE) that was predicted to interact efficiently with VDR-retinoid X receptor α (RXRα) was identified in silico within SLCO1A2 promoter variant 1. This VDRE served as a strong binding site for the recombinant VDR-RXRα heterodimers in vitro and was potently activated by VDR in the presence of vitamin D(3) in heterologous promoter assays. In reporter assays using native promoter constructs, SLCO1A2 promoter variant 1 was strongly induced by VDR, and site-directed mutagenesis of a single VDRE within this region abolished this activation. Native VDR-RXRα also interacted with this element both in vitro and in living cells. We showed that expression of the SLCO1A2 gene is induced by vitamin D(3) at the transcriptional level through the VDR. Our results suggest that pharmacological administration of vitamin D(3) may allow modulation of intestinal absorption of OATP1A2 transport substrates.

Regulation of the gene encoding the intestinal bile acid transporter ASBT by the caudal-type homeobox proteins CDX1 and CDX2.

The apical sodium-dependent bile acid transporter (ASBT) is expressed abundantly in the ileum and mediates bile acid absorption across the apical membranes. Caudal-type homeobox proteins CDX1 and CDX2 are transcription factors that regulate genes involved in intestinal epithelial differentiation and proliferation. Aberrant expression of both ASBT and CDXs in Barrett's esophagus (BE) prompted us to study, whether the expression of the ASBT gene is regulated by CDXs. Short interfering RNA-mediated knockdown of CDXs resulted in reduced ASBT mRNA expression in intestinal cells. CDXs strongly induced the activity of the ASBT promoter in reporter assays in esophageal and intestinal cells. Nine CDX binding sites were predicted in silico within the ASBT promoter, and binding of CDXs to six of them was verified in vitro and within living cells by electrophoretic mobility shift assays and chromatin immunoprecipitation assays, respectively. RNAs were extracted from esophageal biopsies from 20 BE patients and analyzed by real-time PCR. Correlation with ASBT expression was found for CDX1, CDX2, and HNF-1α in BE biopsies. In conclusion, the human ASBT promoter is activated transcriptionally by CDX1 and CDX2. Our finding provides a possible explanation for the reported observation that ASBT is aberrantly expressed in esophageal metaplasia that also expresses CDX transcription factors.

Combined effect of 25-OH vitamin D plasma levels and genetic vitamin D receptor (NR 1I1) variants on fibrosis progression rate in HCV patients.

BACKGROUND: Decreased vitamin D levels have been described in various forms of chronic liver disease and associated with advanced fibrosis. Whether this association is a cause or consequence of advanced fibrosis remains unclear to date. AIMS: To analyse combined effects of 25-OH vitamin D plasma levels and vitamin D receptor gene (VDR; NR1I1) polymorphisms on fibrosis progression rate in HCV patients. METHODS: 251 HCV patients underwent VDR genotyping (bat-haplotype: BsmI rs1544410 C, ApaI rs7975232 A and TaqI rs731236 A). Plasma 25-OH vitamin D levels were quantified in a subgroup of 97 patients without advanced fibrosis. The VDR haplotype and genotypes as well as plasma 25-OH vitamin D levels were associated with fibrosis progression. RESULTS: The bAt[CCA]-haplotype was significantly associated with fibrosis progression >0.101 U/year (P = 0.007; OR = 2.02) and with cirrhosis (P = 0.022; OR = 1.84). Forty-five percent of bAt[CCA]-haplotype patients were rapid fibrosers, 21.1% were cirrhotic. Likewise, ApaI rs7975232 CC genotype was significantly associated with fibrosis progression and cirrhosis. Lower plasma 25-OH vitamin D levels were significantly associated with fibrosis progression >0.101 U/year in F0-2 patients (P = 0.013). Combined analysis of both variables revealed a highly significant additive effect on fibrosis progression with 45.5% rapid fibrosers for bAt[CCA]-haplotype and 25-OH vitamin D < 20 µg/L compared with only 9.1% for the most favourable combination (P = 0.006). In multivariate analysis, the bAt-haplotype was an independent risk factor for fibrosis progression (P = 0.001; OR = 2.83). CONCLUSION: Low 25-OH vitamin D plasma levels and the unfavourable VDR bAt[CCA]-haplotype are associated with rapid fibrosis progression in chronic HCV patients. In combination, both variables exert significant additive effects on fibrosis progression.

Association of a common vitamin D-binding protein polymorphism with inflammatory bowel disease.

OBJECTIVE: Inflammatory bowel diseases (IBDs), Crohn's disease, and ulcerative colitis (UC), are multifactorial disorders, characterized by chronic inflammation of the intestine. A number of genetic components have been proposed to contribute to IBD pathogenesis. In this case-control study, we investigated the association between two common vitamin D-binding protein (DBP) genetic variants and IBD susceptibility. These two single nucleotide polymorphisms (SNPs) in exon 11 of the DBP gene, at codons 416 (GAT>GAG; Asp>Glu) and 420 (ACG>AAG; Thr>Lys), have been previously suggested to play roles in the etiology of other autoimmune diseases. METHODS: Using TaqMan SNP technology, we have genotyped 884 individuals (636 IBD cases and 248 non-IBD controls) for the two DBP variants. RESULTS: On statistical analysis, we observed that the DBP 420 variant Lys is less frequent in IBD cases than in non-IBD controls (allele frequencies, P=0.034; homozygous carrier genotype frequencies, P=0.006). This inverse association between the DBP 420 Lys and the disease remained significant, when non-IBD participants were compared with UC (homozygous carrier genotype frequencies, P=0.022) or Crohn's disease (homozygous carrier genotype frequencies, P=0.016) patients separately. Although the DBP position 416 alone was not found to be significantly associated with IBD, the haplotype DBP_2, consisting of 416 Asp and 420 Lys, was more frequent in the non-IBD population, particularly notably when compared with the UC group (Odds ratio, 4.390). CONCLUSION: Our study adds DBP to the list of potential genes that contribute to the complex genetic etiology of IBD, and further emphasizes the association between vitamin D homeostasis and intestinal inflammation.

The human organic anion transporter genes OAT5 and OAT7 are transactivated by hepatocyte nuclear factor-1α (HNF-1α).

Organic anion transporters (OATs) are anion exchangers that transport small hydrophilic anions and diuretics, antibiotics, nonsteroidal anti-inflammatory drugs, antiviral nucleoside analogs, and antitumor drugs across membrane barriers of epithelia of diverse organs. Three OATs are present in human liver: OAT2, OAT5, and OAT7. Given that hepatocyte nuclear factor-1α (HNF-1α) has previously been shown to regulate the expression of several hepatocellular transporter genes, we investigated whether the liver-specific human OAT genes are also regulated by HNF-1α. Short interfering RNAs targeting HNF-1α reduced endogenous expression of OAT5 and OAT7, but not OAT2, in human liver-derived Huh7 cells. Luciferase reporter gene constructs containing the OAT5 (SLC22A10) and OAT7 (SLC22A9) promoter regions were transactivated by HNF-1α in HepG2 cells. Two putative HNF-1α binding elements in the proximal OAT5 promoter, located at nucleotides -68/-56 and -173/-160, and one element in the OAT7 promoter, located at nucleotides -14/-2 relative to the transcription start site, were shown to bind HNF-1α in electromobility shift assays, and these promoter regions also interacted with HNF-1α in chromatin immunoprecipitation assays. A correlation between HNF-1α and OAT5 (r = 0.134, P < 0.05) or OAT7 (r = 0.461, P < 0.001) mRNA expression levels in surgical liver biopsies from 75 patients further supported an important role of HNF-1α in the regulation of OAT gene expression.

Vitamin D3 and its nuclear receptor increase the expression and activity of the human proton-coupled folate transporter.

Folates are essential for nucleic acid synthesis and are particularly required in rapidly proliferating tissues, such as intestinal epithelium and hemopoietic cells. Availability of dietary folates is determined by their absorption across the intestinal epithelium, mediated by the proton-coupled folate transporter (PCFT) at the apical enterocyte membranes. Whereas transport properties of PCFT are well characterized, regulation of PCFT gene expression remains less elucidated. We have studied the mechanisms that regulate PCFT promoter activity and expression in intestine-derived cells. PCFT mRNA levels are increased in Caco-2 cells treated with 1,25-dihydroxyvitamin D(3) (vitamin D(3)) in a dose-dependent fashion, and the duodenal rat Pcft mRNA expression is induced by vitamin D(3) ex vivo. The PCFT promoter region is transactivated by the vitamin D receptor (VDR) and its heterodimeric partner retinoid X receptor-alpha (RXRalpha) in the presence of vitamin D(3). In silico analyses predicted a VDR response element (VDRE) in the PCFT promoter region -1694/-1680. DNA binding assays showed direct and specific binding of the VDR:RXRalpha heterodimer to the PCFT(-1694/-1680), and chromatin immunoprecipitations verified that this interaction occurs within living cells. Mutational promoter analyses confirmed that the PCFT(-1694/-1680) motif mediates a transcriptional response to vitamin D(3). In functional support of this regulatory mechanism, treatment with vitamin D(3) significantly increased the uptake of [(3)H]folic acid into Caco-2 cells at pH 5.5. In conclusion, vitamin D(3) and VDR increase intestinal PCFT expression, resulting in enhanced cellular folate uptake. Pharmacological treatment of patients with vitamin D(3) may have the added therapeutic benefit of enhancing the intestinal absorption of folates.

Changes in mRNA expression levels of solute carrier transporters in inflammatory bowel disease patients.

Inflammatory bowel disease (IBD) is an inflammatory condition that affects the gastrointestinal tract. The solute carrier (SLC) superfamily of transporters comprise proteins involved in the uptake of drugs, hormones, and other biologically active compounds. The purpose of this study was to determine the mRNA expression levels of 15 solute carrier transporters in two regions of the intestine in IBD patients. Endoscopic biopsy specimens were taken from two locations (terminal ileum and colon) for histological examination and RNA extraction. We quantitatively measured the mRNA expression of 15 SLC transporters in 107 IBD patients (53 with Crohn's disease and 54 with ulcerative colitis) and 23 control subjects. mRNA expression was evaluated using the quantitative reverse transcription-polymerase chain reaction technique. We observed that in the ileum of IBD patients, mRNA levels for serotonin transporter, equilibrative nucleoside transporter (ENT) 1, ENT2, and organic anion-transporting polypeptide (OATP) 2B1 were significantly elevated, whereas levels for apical sodium-dependent bile acid transporter (ASBT) and organic zwitterion/cation transporter (OCTN) 2 were significantly lower. In colon, mRNA levels for ENT1, ENT2, concentrative nucleoside transporter (CNT) 2, OATP2B1, and OATP4A1 were significantly higher, whereas mRNA levels for OCTN2 were significantly decreased. In inflamed colon of IBD patients the mRNA expression levels of ENT1, ENT2, CNT2, OATP2B1, OATP4A1, and peptide transporter 1 were significantly higher. We conclude that intestinal SLC mRNA levels are dysregulated in IBD patients, which may be linked to the inflammation of the tissue and provides an indication about the role of inflammatory signaling in regulation of SLC expression.

The human Na+-taurocholate cotransporting polypeptide gene is activated by glucocorticoid receptor and peroxisome proliferator-activated receptor-gamma coactivator-1alpha, and suppressed by bile acids via a small heterodimer partner-dependent mechanism.

Na+-taurocholate cotransporting polypeptide (NTCP) is the major bile acid uptake system in human hepatocytes. NTCP and the ileal transporter ASBT (apical sodium-dependent bile acid transporter) are two sodium-dependent transporters critical for the enterohepatic circulation of bile acids. The hASBT gene is known to be activated by the glucocorticoid receptor (GR). Here we show that GR also induces the endogenous hNTCP gene and transactivates the reporter-linked hNTCP promoter, in the presence of its ligand dexamethasone. Mutational analysis of the hNTCP promoter identified a functional GR response element, with which GR directly interacts within living cells. The GR/dexamethasone activation of endogenous hNTCP expression was suppressed by bile acids, in a manner dependent on the bile acid receptor farnesoid X receptor. Overexpression of the farnesoid X receptor-inducible transcriptional repressor small heterodimer partner also suppressed the GR/dexamethasone-activation of the hNTCP promoter. The peroxisome proliferator-activated receptor-gamma coactivator-1alpha enhanced the GR/dexamethasone activation of the hNTCP promoter. In conclusion, the hNTCP promoter is activated by GR in a ligand-dependent manner, similarly to the hASBT promoter. Thus, glucocorticoids may coordinately regulate the major bile acid uptake systems in human liver and intestine. The GR/dexamethasone activation of the hNTCP promoter is counteracted by bile acids and small heterodimer partner, providing a negative feedback mechanism for bile acid uptake in human hepatocytes.

Hypoxia Positively Regulates the Expression of pH-Sensing G-Protein-Coupled Receptor OGR1 (GPR68).

BACKGROUND & AIMS: A novel family of proton-sensing G-protein-coupled receptors, including ovarian cancer G-protein-coupled receptor 1 (OGR1) (GPR68) has been identified to play a role in pH homeostasis. Hypoxia is known to change tissue pH as a result of anaerobic glucose metabolism through the stabilization of hypoxia-inducible factor-1α. We investigated how hypoxia regulates the expression of OGR1 in the intestinal mucosa and associated cells. METHODS: OGR1 expression in murine tumors, human colonic tissue, and myeloid cells was determined by quantitative reverse-transcription polymerase chain reaction. The influence of hypoxia on OGR1 expression was studied in monocytes/macrophages and intestinal mucosa of inflammatory bowel disease (IBD) patients. Changes in OGR1 expression in MonoMac6 (MM6) cells under hypoxia were determined upon stimulation with tumor necrosis factor (TNF), in the presence or absence of nuclear factor-κB (NF-κB) inhibitors. To study the molecular mechanisms involved, chromatin immunoprecipitation analysis of the OGR1 promoter was performed. RESULTS: OGR1 expression was significantly higher in tumor tissue compared with normal murine colon tissue. Hypoxia positively regulated the expression of OGR1 in MM6 cells, mouse peritoneal macrophages, primary human intestinal macrophages, and colonic tissue from IBD patients. In MM6 cells, hypoxia-enhanced TNF-induced OGR1 expression was reversed by inhibition of NF-κB. In addition to the effect of TNF and hypoxia, OGR1 expression was increased further at low pH. Chromatin immunoprecipitation analysis showed that HIF-1α, but not NF-κB, binds to the promoter of OGR1 under hypoxia. CONCLUSIONS: The enhancement of TNF- and hypoxia-induced OGR1 expression under low pH points to a positive feed-forward regulation of OGR1 activity in acidic conditions, and supports a role for OGR1 in the pathogenesis of IBD.

Coordinate transcriptional regulation of transport and metabolism.

Intestinal absorption and hepatic clearance of drugs, xenobiotics, and bile acids are mediated by transporter proteins expressed at the plasma membranes of intestinal epithelial cells and liver parenchymal cells in a polarized manner. Within enterocytes and hepatocytes, these exogenous or endogenous, potentially toxic compounds may be metabolized by phase I cytochrome P450 (CYP) and phase II conjugating enzymes. Many transporter proteins and metabolizing enzymes are subject to direct translational modification, enabling very rapid changes in their activity. However, to achieve intermediate and longer term changes in transport and enzyme activities, the genes encoding drug and bile acid transporters, as well as the CYP and conjugating enzymes, are regulated by a complex network of transcriptional cascades. These are typically mediated by specific members of the nuclear receptor family of transcription factors, particularly FXR, SHP, PXR, CAR, and HNF-4alpha. Most nuclear receptors are activated by specific ligands, including numerous xenobiotics (PXR, CAR) and bile acids (FXR). The fine-tuning of transcriptional control of drug and bile acid homeostasis depends on regulated interactions of specific nuclear receptors with their target genes.

Anti-inflammatory Function of High-Density Lipoproteins via Autophagy of IκB Kinase.

BACKGROUND & AIMS: Plasma levels of high-density lipoprotein (HDL) cholesterol are frequently found decreased in patients with inflammatory bowel disease (IBD). Therefore, and because HDL exerts anti-inflammatory activities, we investigated whether HDL and its major protein component apolipoprotein A-I (apoA-I) modulate mucosal inflammatory responses in vitro and in vivo. METHODS: The human intestinal epithelial cell line T84 was used as the in vitro model for measuring the effects of HDL on the expression and secretion of tumor necrosis factor (TNF), interleukin-8 (IL-8), and intracellular adhesion molecule (ICAM). Nuclear factor-κB (NF-κB)-responsive promoter activity was studied by dual luciferase reporter assays. Mucosal damage from colitis induced by dextran sodium sulphate (DSS) and 2,4,6-trinitrobenzenesulfonic acid (TNBS) was scored by colonoscopy and histology in apoA-I transgenic (Tg) and apoA-I knockout (KO) and wild-type (WT) mice. Myeloperoxidase (MPO) activity and TNF and ICAM expression were determined in intestinal tissue samples. Autophagy was studied by Western blot analysis, immunofluorescence, and electron microscopy. RESULTS: HDL and apoA-I down-regulated TNF-induced mRNA expression of TNF, IL-8, and ICAM, as well as TNF-induced NF-κB-responsive promoter activity. DSS/TNBS-treated apoA-I KO mice displayed increased mucosal damage upon both colonoscopy and histology, increased intestinal MPO activity and mRNA expression of TNF and ICAM as compared with WT and apoA-I Tg mice. In contrast, apoA-I Tg mice showed less severe symptoms monitored by colonoscopy and MPO activity in both the DSS and TNBS colitis models. In addition, HDL induced autophagy, leading to recruitment of phosphorylated IκB kinase to the autophagosome compartment, thereby preventing NF-κB activation and induction of cytokine expression. CONCLUSIONS: Taken together, the in vitro and in vivo findings suggest that HDL and apoA-I suppress intestinal inflammation via autophagy and are potential therapeutic targets for the treatment of IBD.

G Protein-coupled pH-sensing Receptor OGR1 Is a Regulator of Intestinal Inflammation.

BACKGROUND: A novel family of proton-sensing G protein-coupled receptors, including OGR1, GPR4, and TDAG8, was identified to be important for physiological pH homeostasis and inflammation. Thus, we determined the function of proton-sensing OGR1 in the intestinal mucosa. MTEHODS: OGR1 expression in colonic tissues was investigated in controls and patients with IBD. Expression of OGR1 upon cell activation was studied in the Mono Mac 6 (MM6) cell line and primary human and murine monocytes by real-time PCR. Ogr1 knockout mice were crossbred with Il-10 deficient mice and studied for more than 200 days. Microarray profiling was performed using Ogr1 and Ogr1 (WT) residential peritoneal macrophages. RESULTS: Patients with IBD expressed higher levels of OGR1 in the mucosa than non-IBD controls. Treatment of MM6 cells with TNF, led to significant upregulation of OGR1 expression, which could be reversed by the presence of NF-κB inhibitors. Kaplan-Meier survival analysis showed a significantly delayed onset and progression of rectal prolapse in female Ogr1/Il-10 mice. These mice displayed significantly less rectal prolapses. Upregulation of gene expression, mediated by OGR1, in response to extracellular acidification in mouse macrophages was enriched for inflammation and immune response, actin cytoskeleton, and cell-adhesion gene pathways. CONCLUSIONS: OGR1 expression is induced in cells of human macrophage lineage and primary human monocytes by TNF. NF-κB inhibition reverses the induction of OGR1 expression by TNF. OGR1 deficiency protects from spontaneous inflammation in the Il-10 knockout model. Our data indicate a pathophysiological role for pH-sensing receptor OGR1 during the pathogenesis of mucosal inflammation.

Functionally significant, rare transcription factor variants in tetralogy of Fallot.

OBJECTIVE: Rare variants in certain transcription factors involved in cardiac development cause Mendelian forms of congenital heart disease. The purpose of this study was to systematically assess the frequency of rare transcription factor variants in sporadic patients with the cardiac outflow tract malformation tetralogy of Fallot (TOF). METHODS AND RESULTS: We sequenced the coding, 5'UTR, and 3'UTR regions of twelve transcription factor genes implicated in cardiac outflow tract development (NKX2.5, GATA4, ISL1, TBX20, MEF2C, BOP/SMYD1, HAND2, FOXC1, FOXC2, FOXH, FOXA2 and TBX1) in 93 non-syndromic, non-Mendelian TOF cases. We also analysed Illumina Human 660W-Quad SNP Array data for copy number variants in these genes; none were detected. Four of the rare variants detected have previously been shown to affect transactivation in in vitro reporter assays: FOXC1 p.P297S, FOXC2 p.Q444R, FOXH1 p.S113T and TBX1 p.P43_G61del PPPPRYDPCAAAAPGAPGP. Two further rare variants, HAND2 p.A25_A26insAA and FOXC1 p.G378_G380delGGG, A488_491delAAAA, affected transactivation in in vitro reporter assays. Each of these six functionally significant variants was present in a single patient in the heterozygous state; each of the four for which parental samples were available were maternally inherited. Thus in the 93 TOF cases we identified six functionally significant mutations in the secondary heart field transcriptional network. SIGNIFICANCE: This study indicates that rare genetic variants in the secondary heart field transcriptional network with functional effects on protein function occur in 3-13% of patients with TOF. This is the first report of a functionally significant HAND2 mutation in a patient with congenital heart disease.

Identification of a SIRT1 mutation in a family with type 1 diabetes.

Type 1 diabetes is caused by autoimmune-mediated ß cell destruction leading to insulin deficiency. The histone deacetylase SIRT1 plays an essential role in modulating several age-related diseases. Here we describe a family carrying a mutation in the SIRT1 gene, in which all five affected members developed an autoimmune disorder: four developed type 1 diabetes, and one developed ulcerative colitis. Initially, a 26-year-old man was diagnosed with the typical features of type 1 diabetes, including lean body mass, autoantibodies, T cell reactivity to ß cell antigens, and a rapid dependence on insulin. Direct and exome sequencing identified the presence of a T-to-C exchange in exon 1 of SIRT1, corresponding to a leucine-to-proline mutation at residue 107. Expression of SIRT1-L107P in insulin-producing cells resulted in overproduction of nitric oxide, cytokines, and chemokines. These observations identify a role for SIRT1 in human autoimmunity and unveil a monogenic form of type 1 diabetes.

The Impact of PPARγ Genetic Variants on IBD Susceptibility and IBD Disease Course.

PPARγ is a nuclear receptor that regulates numerous pathways including cytokine expression and immune responses and plays an important role in controlling colon inflammation. We aimed at determining the occurring PPARγ SNPs, at predicting the haplotypes, and at determining the frequency outcome in inflammatory bowel disease (IBD) patients in comparison with healthy controls. We determined genetic variants in the coding exons and flanking intronic sequences of the NR1C3 gene in 284 IBD patients and 194 controls and predicted NR1C3 haplotypes via bioinformatic analysis. We investigated whether certain NR1C3 variants are associated with susceptibility to IBD or its disease course. None of the detected 22 NR1C3 variants were associated with IBD. Two variants with allelic frequencies over 1% were included in haplotype/diplotype analyses. None of the NR3C1 haplotypes showed association with IBD development or disease course. We conclude that NR1C3 haplotypes are not related to IBD susceptibility or IBD disease activity.

Association of genetic variation in the NR1H4 gene, encoding the nuclear bile acid receptor FXR, with inflammatory bowel disease.

BACKGROUND: Pathogenesis of inflammatory bowel diseases (IBD), ulcerative colitis (UC) and Crohn's disease (CD), involves interaction between environmental factors and inappropriate immune responses in the intestine of genetically predisposed individuals. Bile acids and their nuclear receptor, FXR, regulate inflammatory responses and barrier function in the intestinal tract. METHODS: We studied the association of five variants (rs3863377, rs7138843, rs56163822, rs35724, rs10860603) of the NR1H4 gene encoding FXR with IBD. 1138 individuals (591 non-IBD, 203 UC, 344 CD) were genotyped for five NR1H4 genetic variants with TaqMan SNP Genotyping Assays. RESULTS: We observed that the NR1H4 SNP rs3863377 is significantly less frequent in IBD cases than in non-IBD controls (allele frequencies: P = 0.004; wild-type vs. SNP carrier genotype frequencies: P = 0.008), whereas the variant rs56163822 is less prevalent in non-IBD controls (allele frequencies: P = 0.027; wild-type vs. SNP carrier genotype frequencies: P = 0.035). The global haplotype distribution between IBD and control patients was significantly different (P = 0.003). This also held true for the comparison between non-IBD and UC groups (P = 0.004), but not for the comparison between non-IBD and CD groups (P = 0.079). CONCLUSIONS: We show that genetic variation in FXR is associated with IBD, further emphasizing the link between bile acid signaling and intestinal inflammation.

Fc gamma receptor CD64 modulates the inhibitory activity of infliximab.

BACKGROUND: Tumor necrosis factor (TNF) is an important cytokine in the pathogenesis of inflammatory bowel disease (IBD). Anti-TNF antibodies have been successfully implemented in IBD therapy, however their efficacies differ among IBD patients. Here we investigate the influence of CD64 Fc receptor on the inhibitory activity of anti-TNFs in cells of intestinal wall. METHODS: Intestinal cell lines, monocytes/macrophages and peripheral blood mononuclear cells (PBMCs) were used as models. The efficacies of adalimumab, infliximab and certolizumab-pegol were assessed by RT-PCR for target genes. Protein levels and localizations were examined by Western blotting and immunofluorescence. Antibody fragments were obtained by proteolytic digestion, immunoprecipitation and protein chip analysis. Knock-down of specific gene expression was performed using siRNAs. RESULTS: Infliximab had limited efficacy towards soluble TNF in cell types expressing Fc gamma receptor CD64. Both adalimumab and infliximab had lower efficacies in PBMCs of IBD patients, which express elevated levels of CD64. Infliximab-TNF complexes were more potent in activating CD64 in THP-1 cells than adalimumab, which was accompanied by distinct phospho-tyrosine signals. Blocking Fc parts and isolation of Fab fragments of infliximab improved its efficacy. IFN-γ-induced expression of CD64 correlated with a loss of efficacy of infliximab, whereas reduction of CD64 expression by either siRNA or PMA treatment improved inhibitory activity of this drug. Colonic mRNA expression levels of CD64 and other Fc gamma receptors were significantly increased in the inflamed tissues of infliximab non-responders. CONCLUSIONS: CD64 modulates the efficacy of infliximab both in vitro and ex vivo, whereas the presence of this receptor has no impact on the inhibitory activity of certolizumab-pegol, which lacks Fc fragment. These data could be helpful in both predicting and evaluating the outcome of anti-TNF therapy in IBD patients with elevated systemic and local levels of Fc receptors.