Functional assessment of genetic variants located in the promoter of SHP1 (NR0B2)

Small heterodimer partner 1 (SHP1, NR0B2) is a member of the superfamily of nuclear receptors (NRs). Even if this orphan receptor, unlike other NRs, lacks the DNA-binding domain, it is capable of regulating transcription by repressing the activity of other NRs by direct protein-protein interaction. Accordingly, SHP1 is part of negative feedback loops of the transcriptional regulation of genes involved in drug metabolism and various metabolic pathways including bile acid and glucose homeostasis. Although it is known that several interacting partners of SHP1 also modulate its expression, there is little information about genetic variability of this regulatory mechanism. Our study aimed to identify genetic variants in the NR0B2 promoter region and to determine their impact on NR0B2 transcription. For this, DNA samples originating from 119 participants of the population-based cohort Study of Health in Pomerania were analyzed by Sanger sequencing revealing four genetic variants: NR0B2:c.-594T>C (rs71636795), NR0B2:c.-414G>C (newly identified), NR0B2:c.-423C>T (rs78182695), and NR0B2:c.-224delCTGA (rs145613139) localized in the 5' untranslated region of NR0B2. The impact of these variants on transactivation of the NR0B2 promoter by NRs known to be regulators of SHP1 expression (hepatocyte nuclear factor 4α, liver receptor homolog-1, and farnesoid X receptor) was assessed in a cell-based reporter gene assay, showing that transactivation by hepatocyte nuclear factor 4α and liver receptor homolog-1 was significantly decreased in the presence of the genetic variant NR0B2:c.-594T>C, even though this effect was cell specific. However, SHP1 mRNA expression in a small collection of human kidney samples was not affected by these genetic variants

PDZ domain containing protein 1 (PDZK1), a modulator of membrane proteins, is regulated by the nuclear receptor THRβ

Genome wide association studies revealed single nucleotide polymorphisms (SNP) located within the promoter of PDZ domain containing protein 1 (PDZK1) to be associated with serum uric acid levels. Since modulation of transporters and particularly of membrane proteins involved in uric acid handling by PDZK1 has previously been reported, the aim of this study was to analyze the impact of the polymorphisms rs1967017, rs1471633, and rs12129861 on promoter activity and thereby transcription of PDZK1. Cell-based reporter gene assays showed transactivation of the PDZK1-promoter by triiodothyronine mediated by thyroid hormone receptors (THR) α and β. In silico analysis verified localization of the polymorphism rs1967017 within the most likely THR binding site whose deletion reduced THR-mediated transactivation. Furthermore, our study shows regulation of PDZK1 by thyroid hormones, thereby providing a mechanistic basis for the previously reported associations between thyroid hormone status and uric acid homeostasis

PDMS-PMOXA-Nanoparticles Featuring a Cathepsin B-Triggered Release Mechanism

It was our intention to develop cathepsin B-sensitive nanoparticles for tumor-site-directed release. These nanoparticles should be able to release their payload as close to the tumor site with a decrease of off-target effects in mind. Cathepsin B, a lysosomal cysteine protease, is associated with premalignant lesions and invasive stages of cancer. Previous studies have shown cathepsin B in lysosomes and in the extracellular matrix. Therefore, this enzyme qualifies as a trigger for such an approach.; Poly(dimethylsiloxane)-b-poly(methyloxazoline) (PDMS-PMOXA) nanoparticles loaded with paclitaxel were formed by a thin-film technique and standard coupling reactions were used for surface modifications. Despite the controlled release mechanism, the physical properties of the herein created nanoparticles were described. To characterize potential in vitro model systems, quantitative polymerase chain reaction and common bioanalytical methods were employed.; Stable paclitaxel-loaded nanoparticles with cathepsin B digestible peptide were formed and tested on the ovarian cancer cell line OVCAR-3. These nanoparticles exerted a pharmacological effect on the tumor cells suggesting a release of the payload

OATP1B3-1B7 a novel Organic Anion Transporting Polypeptide is modulated by FXR ligands and transports bile acids

OATP1B3-1B7 (LST-3TM12) is a member of the OATP1B (; SLCO1B; )-family. This transporter is not only functional, but also expressed in the membrane of the smooth endoplasmic reticulum of hepatocytes and enterocytes. OATP1B3-1B7 is a splice variant of; SLCO1B3; where the initial part is encoded by; SLCO1B3; , whereas the rest of the mRNA originates from the gene locus of; SLCO1B7; . In this study we not only showed that; SLCO1B3; and the mRNA encoding for OATP1B3-1B7 share the 5' untranslated region, but also that silencing of an initial; SLCO1B3; exon lowered the amount of; SLCO1B3; and of; SLCO1B7; mRNA in Huh-7 cells. To validate the assumption that both transcripts are regulated by the same promoter we tested the influence of the bile acid sensor farnesoid X receptor (FXR) on their transcription. Treatment of Huh-7 and HepaRG cells with activators of this known regulator of OATP1B3 not only increased; SLCO1B3,; but also OATP1B3-1B7 mRNA transcription. Applying a heterologous expression system we showed that several bile acids interact with OATP1B3-1B7 and that taurocholic acid and lithocholic acid are OATP1B3-1B7 substrates. As OATP1B3-1B7 is located in the smooth endoplasmic reticulum it may grant access to metabolizing enzymes. In accordance are our findings showing that the OATP1B3-1B7 inhibitor bromsulphthalein significantly reduced uptake of bile acids into human liver microsomes. Taken together we report that OATP1B3-1B7 transcription can be modulated with FXR agonists and antagonists and that OATP1B3-1B7 transports bile acids

Pimecrolimus increases the expression of interferon-inducible genes that modulate human coronary artery cells proliferation

The pharmacodynamics of the loaded compounds defines clinical failure or success of a drug-eluting device. Various limus derivatives have entered clinics due to the observed positive outcome after stent implantation, which is explained by their antiproliferative activity resulting from inhibition of the cytosolic immunophilin FK506-binding protein 12. Although pimecrolimus also binds to this protein, pimecrolimus-eluting stents failed in clinics. However, despite its impact on T lymphocytes little is known about the pharmacodynamics of pimecrolimus in cultured human coronary artery cells. We were able to show that pimecrolimus exerts antiproliferative activity in human smooth muscle and endothelial cells. Furthermore in those cells pimecrolimus induced transcription of interferon-inducible genes which in part are known to modulate cell proliferation. Modulation of gene expression may be part of an interaction between calcineurin, the downstream target of the pimecrolimus/FK506-binding protein 12-complex, and the toll-like receptor 4. In accordance are our findings showing that silencing of toll-like receptor 4 by siRNA in A549 a lung carcinoma cell line reduced the activation of interferon-inducible genes upon pimecrolimus treatment in those cells. Based on our findings we hypothesize that calcineurin inhibition may induce the toll-like receptor 4 mediated activation of type I interferon signaling finally inducing the observed effect in endothelial and smooth muscle cells. The crosstalk of interferon and toll-like receptor signaling may be a molecular mechanism that contributed to the failure of pimecrolimus-eluting stents in humans

Expression of OATP2B1 as determinant of drug effects in the microcompartment of the coronary artery

Clinical success of coronary drug-eluting stents (DES) is hampered by simultaneous reduction of smooth muscle cell (HCASMC) and endothelial cell proliferation due to unspecific cytotoxicity of currently used compounds. Previous in vitro data showing SMC-specific inhibition of proliferation suggested that statins may be suitable candidates for DES. It was aim of this study to further investigate statins as DES drug candidates to identify mechanisms contributing to their cell-selectivity. In vitro proliferation assays comparing the influence of various statins on HCASMC and endothelial cells confirmed that atorvastatin exhibits HCASMC-specificity. Due to similar expression levels of the drug target HMG-CoA reductase in both cell types, cellular accumulation of atorvastatin was assessed, revealing enhanced uptake in HCASMC most likely driven by significant expression of OATP2B1, a known uptake transporter for atorvastatin. In accordance with the finding that endogenous OATP2B1 influenced cellular accumulation in HCASMC we used this transporter as a tool to identify teniposide as new DES candidate drug with HCASMC-specific effects. We describe OATP2B1 as a determinant of pharmacokinetics in the coronary artery. Indeed, endogenously expressed OATP2B1 significantly influences the uptake of substrate drugs, thereby governing cell specificity. Screening of candidate drugs for interaction with OATP2B1 may be used to promote SMC-specificity

LST-3TM12 is a member of the OATP1B family and a functional transporter

Organic anion transporting polypeptides (OATPs) and particularly the two members of the OATP1B family are known for their role in pharmacokinetics. Both SLCO1B3 and SLCO1B1 are located on chromosome 12 encompassing the gene locus SLCO1B7. Hitherto, this particular gene has been assumed to be a pseudogene, even though there are published mRNA sequences linked to this chromosomal area. It was aim of this study to further investigate SLCO1B7 and the associated mRNA LST-3TM12. In a first step, we aligned all mRNAs linked to the chromosomal region of SLCO1B-transporters. This in silico analysis revealed that LST-3TM12 is a product of splicing of SLCO1B3 and SLCO1B7, and encodes for a protein with twelve transmembrane domains. The existence of LST-3TM12 mRNA was verified by polymerase chain reaction showing liver enriched expression. In addition, immunohistological staining showed that LST-3TM12 protein was expressed in the endoplasmic reticulum (ER) of hepatocytes. Localization in the ER was further verified by immunoblot analysis showing high amounts of LST-3TM12 in liver microsomes. Function of LST-3TM12 was assessed by transport studies after heterologous expression in HeLa cells, where the transporter was shown to be expressed not only in the ER but also in the plasma membrane. Overexpression of LST-3TM12 was associated with enhanced cellular accumulation of dehydroepiandrosterone sulfate (Vmax 300.2pmol mg-1 min-1; Km 34.2µm) and estradiol 17β-glucuronide (Vmax 29.9mol mg-1 min-1 and Km 32.8µM). In conclusion, LST-3TM12 is a functional splice variant of SLCO1B3 and SLCO1B7 expressed in the ER of human liver

Regulation of PDZ domain containing 1 (PDZK1) Expression by Hepatocyte Nuclear Factor 1 alpha (HNF1α) in Human Kidney

In the renal proximal tubule the secretion and reabsorption of glomerularly filtrated compounds is realized by a functional network of uptake and efflux transporters. The activity and localization of several transporters expressed at the apical tubular membrane is regulated by the membrane associated protein PDZ domain containing 1 (PDZK1). We aimed to characterize the transcriptional regulation of this modulator of renal transport. Coexpression analyses of PDZK1 and putative regulators were performed using human kidney samples. Protein and mRNA expression of PDZK1 in renal proximal tubule epithelial cells after adenoviral transfer and siRNA knockdown of transcription factor hepatocyte nuclear factor 1 alpha (HNF1α) was assessed by quantitative real-time PCR and Western blot analysis. Transactivation of the PDZK1 promoter was quantified in cell-based reporter gene assays. Subsequently, the binding of HNF1α to the PDZK1 promoter was verified by in silico analyses and chromatin immunoprecipitation assay. HNF1α positively regulated the promoter activity of PDZK1. Adenoviral overexpression of HNF1α in renal proximal tubule epithelial cells (RPTEC) increased PDZK1 mRNA and protein expression, whereas siRNA knockdown of HNF1α resulted in decreased expression of PDZK1. Our results show that HNF1α, which has previously been described as a modulator of several transporters of the renal transportosome, is also a key determinant of PDZK1 transcription

The scaffold protein PDZK1 modulates expression and function of the organic anion transporting polypeptide 2B1

The protein family of Organic Anion Transporting Polypeptides (OATPs) summarizes various transporters known to facilitate cellular uptake of xenobiotics. One member of this family is OATP2B1. This transporter is ubiquitously expressed and possesses a PDZ-binding motif at the C-terminus. PDZK1 (PDZ domain-containing 1) is a scaffold protein that influences function of different membrane proteins by sorting/stabilization of their membrane localization. It was aim of the herein reported study to investigate whether there is an interaction between OATP2B1 and PDZK1, and to further characterize its impact on transport function. At first expression of both OATP2B1 and PDZK1 was evaluated in liver, kidney and intestine. Based on the existence of a C-terminal PDZ-class I binding motif in OATP2B1 and the co-expression in all tested tissues an interaction was likely. Testing the influence of PDZK1 on OATP2B1 transport function revealed enhanced transport capacity for estrone 3-sulfate, thereby suggesting a change in OATP2B1 amount in the membrane. This assumption was validated by Western blot analysis. Finally, deletion of the C-terminal PDZ-binding motif in OATP2B1 lowered the impact of PDZK1 on transport function. Taken together, we report an interaction of PDZK1 with OATP2B1, which influences localization and function of the transporter. Changes in PDZK1 expression may therefore be one factor contributing to interindividual differences in OATP2B1 mediated pharmacokinetic processes