Jyrki J. Eloranta

Regional Distribution of Solute Carrier mRNA Expression Along the Human Intestinal Tract

Intestinal absorption of drugs, nutrients, and other compounds is mediated by uptake transporters expressed at the apical enterocyte membrane. These compounds are returned to the intestinal lumen or released into portal circulation by intestinal efflux transporters expressed at apical or basolateral membranes, respectively. One important transporter superfamily, multiple members of which are intestinally expressed, are the solute carriers (SLCs). SLC expression levels may determine the pharmacokinetics of drugs that are substrates of these transporters. In this study we characterize the distribution of 15 human SLC transporter mRNAs in histologically normal biopsies from five regions of the intestine of 10 patients. The mRNA expression levels of CNT1, CNT2, apical sodium-dependent bile acid transporter (ABST), serotonin transporter (SERT), PEPT1, and OCTN2 exhibit marked differences between different regions of the intestine: the first five are predominantly expressed in the small intestine, whereas OCTN2 exhibits strongest expression in the colon. Two transporter mRNAs studied (OCTN1, OATP2B1) are expressed at similar levels in all gut sections. In addition, ENT2 mRNA is present at low levels across the colon, but not in the small intestine. The other six SLC mRNAs studied are not expressed in the intestine. Quantitative knowledge of transporter expression levels in different regions of the human gastrointestinal tract could be useful for designing intestinal delivery strategies for orally administered drugs. Furthermore, changes in transporter expression that occur in pathological states, such as inflammatory bowel disease, can now be defined more precisely by comparison with the expression levels measured in healthy individuals.

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 Crohns 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.

Pharmacogenetics of OATP (SLC21/SLCO), OAT and OCT (SLC22) and PEPT (SLC15) transporters in the intestine, liver and kidney

The role of carrier-mediated transport in determining the pharmacokinetics of drugs has become increasingly evident with the discovery of genetic variants that affect expression and/or function of a given drug transporter. Drug transporters are expressed at numerous epithelial barriers, such as intestinal epithelial cells, hepatocytes, renal tubular cells and at the blood-brain barrier. Several recent studies have associated alterations in substrate uptake with the presence of SNPs. Here, we summarize the current knowledge on the functional and phenotypic consequences of genetic variation in intestinally, hepatically and renally expressed members of the organic anion-transporting polypeptide family (OATPs; SLC21/SLCO family), the organic anion and organic cation transporters (OATs/OCTs; SLC22 family) and the peptide transporter-1 (PEPT1; SLC15 family).

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

Type 1 diabetes is caused by autoimmune-mediated β 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 β 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.

Squamous cell carcinoma of the skin shows a distinct microRNA profile modulated by UV radiation.

investigated CHR expression in mast cells just in vicinity of eccrine glands. Serial sections were alternatively stained with toluidine blue and with anti-CHRM3 antibody. Mast cells were identified by positive toluidine blue staining (Figure 2c, upper panel), and CHRM3 expression by mast cells was examined in the antibody-stained adjacent sections (Figure 2c, lower panel). In a normal control, mast cells expressed CHRM3 at high levels. Mast cells in conjunction with the secretory portion expressed CHRM3 in the hypohidrotic but not anhidrotic areas. There was no significant difference in the number of mast cells between the anhidrotic and hypohidrotic areas. Our study revealed that the skin of patients with CUAH is divided into the wheal-non-occuring anhidrotic and wheal-occuring hypohidrotic areas. Even in the hypohidrotic areas, the intradermal injection of autologous sweat did not yield wheal, suggesting the absence of sweat allergy (Tsuchiya et al., 2004). We found the lack of CHRM3 expression in the anhidrotic skin, which may lead to the lack of sensitivity to acetylcholine. Mast cells are responsible for wheal formation and present just in the vicinity of eccrine glands. Neither eccrine gland cells nor mast cells expressed CHRM3 in the anhidrotic area, and it is thus reasonable that sweating and wheal formation were absent in this area. CHR mediates wheal development (Tong et al., 1997), and acetylcholine can induce degranulation of mast cells (Fantozzi et al., 1978; Blandina et al., 1980). In the hypohidrotic area, we are tempting to speculate that acetylcholine released from nerves upon exercise cannot be completely trapped by CHR of eccrine glands and overflows to the adjacent mast cells. In this scenario, mast cells may be capable of producing histamine and resultant wheal in response to acetylcholine because of the expression of some degree of CHRM3.

The Human Organic Cation Transporter-1 Gene Is Transactivated by Hepatocyte Nuclear Factor-4α

The organic cation transporter-1 (OCT1) mediates the hepatocellular uptake of cationic drugs and endobiotics from sinusoidal blood. The uptake rates of these compounds may depend on OCT1 expression level. Because little is known about the regulation of the human OCT1 (hOCT1) gene, we characterized the hOCT1 promoter with respect to DNA-response elements and their binding factors. By computer analysis, we identified two adjacent putative DNA-response elements for the liver-enriched homodimeric nuclear receptor hepatocyte nuclear factor-4α (HNF-4α) in the hOCT1 promoter. Each element is of the direct repeat (DR)-2 format, containing directly repeated hexamers separated by two bases. In electrophoretic mobility shift assays, both elements directly interacted with HNF-4α. A luciferase reporter construct containing the hOCT1 promoter was strongly activated by HNF-4α in transiently transfected Huh7 cells. Site-directed mutagenesis of either DR-2 element alone or in combination severely decreased the HNF-4α-mediated activation of the hOCT1 promoter, indicating that both elements are functionally important. Because HNF-4α is a known target for bile acid-mediated suppression of transcription, we studied whether chenodeoxycholic acid (CDCA) suppresses hOCT1 gene expression by inhibiting HNF-4α-mediated transactivation. Treatment of cells with CDCA could indeed suppress the activation of the endogenous hOCT1 gene by HNF-4α. In addition, bile acid-inducible transcriptional repressor, small heterodimer partner (SHP), inhibited activation of the reporter-linked hOCT1 promoter and of the endogenous hOCT1 gene by HNF-4α. In conclusion, the hOCT1 gene, encoding an important drug transporter in the human liver, is activated by HNF-4α and suppressed by bile acids via SHP.

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

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.

The Role of FXR in Disorders of Bile Acid Homeostasis

As ligands for the nuclear receptor FXR, bile acids regulate their own synthesis, transport, and conjugation, thus protecting against bile acid toxicity. Recently, the role of genetic variants in FXR itself, FXR target genes, and regulators of FXR in the pathophysiology of the liver and intestine has become increasingly evident.

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.

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 D3 (vitamin D3) in a dose-dependent fashion, and the duodenal rat Pcft mRNA expression is induced by vitamin D3 ex vivo. The PCFT promoter region is transactivated by the vitamin D receptor (VDR) and its heterodimeric partner retinoid X receptor-α (RXRα) in the presence of vitamin D3. 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:RXRα 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 D3. In functional support of this regulatory mechanism, treatment with vitamin D3 significantly increased the uptake of [3H]folic acid into Caco-2 cells at pH 5.5. In conclusion, vitamin D3 and VDR increase intestinal PCFT expression, resulting in enhanced cellular folate uptake. Pharmacological treatment of patients with vitamin D3 may have the added therapeutic benefit of enhancing the intestinal absorption of folates.