A TNFRSF14-FcɛRI-mast cell pathway contributes to development of multiple features of asthma pathology in mice.

Asthma has multiple features, including airway hyperreactivity, inflammation and remodelling. The TNF superfamily member TNFSF14 (LIGHT), via interactions with the receptor TNFRSF14 (HVEM), can support TH2 cell generation and longevity and promote airway remodelling in mouse models of asthma, but the mechanisms by which TNFSF14 functions in this setting are incompletely understood. Here we find that mouse and human mast cells (MCs) express TNFRSF14 and that TNFSF14:TNFRSF14 interactions can enhance IgE-mediated MC signalling and mediator production. In mouse models of asthma, TNFRSF14 blockade with a neutralizing antibody administered after antigen sensitization, or genetic deletion of Tnfrsf14, diminishes plasma levels of antigen-specific IgG1 and IgE antibodies, airway hyperreactivity, airway inflammation and airway remodelling. Finally, by analysing two types of genetically MC-deficient mice after engrafting MCs that either do or do not express TNFRSF14, we show that TNFRSF14 expression on MCs significantly contributes to the development of multiple features of asthma pathology.

Clinical and pharmacogenetic predictors of circulating atorvastatin and rosuvastatin concentrations in routine clinical care.

BACKGROUND: A barrier to statin therapy is myopathy associated with elevated systemic drug exposure. Our objective was to examine the association between clinical and pharmacogenetic variables and statin concentrations in patients. METHODS AND RESULTS: In total, 299 patients taking atorvastatin or rosuvastatin were prospectively recruited at an outpatient referral center. The contribution of clinical variables and transporter gene polymorphisms to statin concentration was assessed using multiple linear regression. We observed 45-fold variation in statin concentration among patients taking the same dose. After adjustment for sex, age, body mass index, ethnicity, dose, and time from last dose, SLCO1B1 c.521T>C (P<0.001) and ABCG2 c.421C>A (P<0.01) were important to rosuvastatin concentration (adjusted R(2)=0.56 for the final model). Atorvastatin concentration was associated with SLCO1B1 c.388A>G (P<0.01) and c.521T>C (P<0.05) and 4ß-hydroxycholesterol, a CYP3A activity marker (adjusted R(2)=0.47). A second cohort of 579 patients from primary and specialty care databases were retrospectively genotyped. In this cohort, genotypes associated with statin concentration were not differently distributed among dosing groups, implying providers had not yet optimized each patient's risk-benefit ratio. Nearly 50% of patients in routine practice taking the highest doses were predicted to have statin concentrations greater than the 90th percentile. CONCLUSIONS: Interindividual variability in statin exposure in patients is associated with uptake and efflux transporter polymorphisms. An algorithm incorporating genomic and clinical variables to avoid high atorvastatin and rosuvastatin levels is described; further study will determine whether this approach reduces incidence of statin myopathy.

Interaction of three regiospecific amino acid residues is required for OATP1B1 gain of OATP1B3 substrate specificity.

The human organic anion-transporting polypeptides OATP1B1 (SLCO1B1) and OATP1B3 (SLCO1B3) are liver-enriched membrane transporters of major importance to hepatic uptake of numerous endogenous compounds, including bile acids, steroid conjugates, hormones, and drugs, including the 3-hydroxy-3-methylglutaryl Co-A reductase inhibitor (statin) family of cholesterol-lowering compounds. Despite their remarkable substrate overlap, there are notable exceptions: in particular, the gastrointestinal peptide hormone cholecystokinin-8 (CCK-8) is a high affinity substrate for OATP1B3 but not OATP1B1. We utilized homologous recombination of linear DNA by E. coli to generate a library of cDNA containing monomer size chimeric OATP1B1-1B3 and OATP1B3-1B1 transporters with randomly distributed chimeric junctions to identify three discrete regions of the transporter involved in conferring CCK-8 transport activity. Site-directed mutagenesis of three key residues in OATP1B1 transmembrane helices 1 and 10, and extracellular loop 6, to the corresponding residues in OATP1B3, resulted in a gain of CCK-8 transport by OATP1B1. The residues appear specific to CCK-8, as the mutations did not affect transport of the shared OATP1B substrate atorvastatin or the OATP1B1-specific substrate estrone sulfate. Regions involved in gain of CCK-8 transport by OATP1B1, when mapped to the crystal structures of bacterial transporters from the major facilitator superfamily, are positioned to suggest these regions could readily interact with drug substrates. Accordingly, our data provide new insight into the molecular determinants of the substrate specificity of these hepatic uptake transporters with relevance to targeted drug design and prediction of drug-drug interactions.

Structural determinants of substrate specificity differences between human multidrug resistance protein (MRP) 1 (ABCC1) and MRP3 (ABCC3).

Multidrug resistance proteins (MRPs) are members of the "C" branch of the ATP-binding cassette transporter superfamily. Human MRP1 transports a wide range of natural product drugs and structurally diverse conjugated and unconjugated organic anions. Its closest relative is MRP3. Despite their structural similarity, the homologs differ substantially in their substrate specificity. It is noteworthy that MRP1 transports glutathione (GSH) and GSH conjugates and displays GSH-stimulated transport of a number of unconjugated and conjugated compounds. In contrast, MRP3 does not transport GSH and is a poor transporter of GSH conjugates. However, both proteins transport glucuronide conjugates, such as 17beta-estradiol 17-(beta-D-glucuronide). We have constructed a series of MRP1/MRP3 hybrids and used them to identify a region of MRP1 that is critical for binding and transport of GSH conjugates such as leukotriene C(4) (LTC(4)). Substitution of this region encompassing transmembrane helices 8 and 9 and portions of cytoplasmic loops 4 and 5 of MRP1 with the equivalent region of MRP3 eliminated LTC(4) transport. Transport of other substrates was either unaffected or enhanced. We identified three residues in this region: Tyr(440), Ile(441), and Met(443), mutation of which differentially affected transport. It is noteworthy that substitution of Tyr(440) with Phe, as found in MRP3, reduced LTC(4) and GSH-stimulated estrone-3-sulfate transport without affecting transport of other substrates tested. The mutation increased the K(m) for LTC(4) 5-fold and substantially reduced photolabeling of MRP1 by both [3H]LTC(4) and the GSH derivative, azidophenacyl-[35S]GSH. These results suggest that Tyr(440) makes a major contribution to recognition of GSH and the GSH moiety of conjugates such as LTC(4).

Molecular modeling of the human multidrug resistance protein 1 (MRP1/ABCC1).

Multidrug resistance protein 1 (MRP1/ABCC1) is a 190kDa member of the ATP-binding cassette (ABC) superfamily of transmembrane transporters that is clinically relevant for its ability to confer multidrug resistance by actively effluxing anticancer drugs. Knowledge of the atomic structure of MRP1 is needed to elucidate its transport mechanism, but only low resolution structural data are currently available. Consequently, comparative modeling has been used to generate models of human MRP1 based on the crystal structure of the ABC transporter Sav1866 from Staphylococcus aureus. In these Sav1866-based models, the arrangement of transmembrane helices differs strikingly from earlier models of MRP1 based on the structure of the bacterial lipid transporter MsbA, both with respect to packing of the twelve helices and their interactions with the nucleotide binding domains. The functional importance of Tyr324 in transmembrane helix 6 predicted to project into the substrate translocation pathway was investigated.