Endotoxin leads to rapid subcellular re-localization of hepatic RXRα: A novel mechanism for reduced hepatic gene expression in inflammation

BACKGROUND: Lipopolysaccharide (LPS) treatment of animals down-regulates the expression of hepatic genes involved in a broad variety of physiological processes, collectively known as the negative hepatic acute phase response (APR). Retinoid X receptor α (RXRα), the most highly expressed RXR isoform in liver, plays a central role in regulating bile acid, cholesterol, fatty acid, steroid and xenobiotic metabolism and homeostasis. Many of the genes regulated by RXRα are repressed during the negative hepatic APR, although the underlying mechanism is not known. We hypothesized that inflammation-induced alteration of the subcellular location of RXRα was a common mechanism underlying the negative hepatic APR. RESULTS: Nuclear RXRα protein levels were significantly reduced (~50%) within 1–2 hours after low-dose LPS treatment and remained so for at least 16 hours. RXRα was never detected in cytosolic extracts from saline-treated mice, yet was rapidly and profoundly detectable in the cytosol from 1 hour, to at least 4 hours, after LPS administration. These effects were specific, since the subcellular localization of the RXRα partner, the retinoic acid receptor (RARα), was unaffected by LPS. A potential cell-signaling modulator of RXRα activity, c-Jun-N-terminal kinase (JNK) was maximally activated at 1–2 hours, coincident with maximal levels of cytoplasmic RXRα. RNA levels of RXRα were unchanged, while expression of 6 sentinel hepatic genes regulated by RXRα were all markedly repressed after LPS treatment. This is likely due to reduced nuclear binding activities of regulatory RXRα-containing heterodimer pairs. CONCLUSION: The subcellular localization of native RXRα rapidly changes in response to LPS administration, correlating with induction of cell signaling pathways. This provides a novel and broad-ranging molecular mechanism for the suppression of RXRα-regulated genes in inflammation

Altered Irinotecan Pharmacokinetics in Diet-Induced Obesity

Title: Altered Irinotecan Pharmacokinetics in Diet-Induced Obesity Authors: Pranav Shah*, Adarsh Gandhi and Romi Ghose Affiliation: University of Houston, Department of Pharmacological and Pharmaceutical Sciences, Houston, TX - 77030 Purpose: Irinotecan (CPT-11) is a topoisomerase I inhibitor that has been shown to be highly effective in treatment of colon, stomach, pancreas, and non-small cell lung cancers. It has recently been shown that CPT-11 administration is associated with liver toxicity and this effect is compounded by baseline obesity. It was found that patients with a BMI index of \u3e25 were twice as much susceptible to developing liver toxicity than patients with BMI index of Methods: For drug metabolism studies, liver S9 fractions were prepared from diet-induced obese (DIO, 60% Kcal diet fed mice) and lean mice (10% Kcal diet fed mice). UGT1A-mediated metabolism of SN-38 was determined in liver S9 fractions (2 mg/ml protein) incubated with SN-38 (15 µmol) for 60 min. For pharmacokinetic studies, mice were injected with a single oral dose of 10 mg/kg CPT-11 and blood samples were collected from 0-480 minutes. Plasma and feces samples were analyzed for CPT-11 and SN-38 concentrations using LC-MS/MS. Liver tissues were harvested for real-time PCR studies. The mRNA and serum TNF-α levels were measured in liver and plasma samples, respectively. Results: We found that the rate of formation of SN-38G was ~2 fold lower in the DIO mice compared to the lean controls. This corresponded with reduced expression of UGT1A1 in DIO mice livers. We did not observe significant changes in the area under the curve (AUC) or clearance of CPT-11 between the DIO and lean mice. However, plasma and fecal exposure (AUC) of SN-38 was increased by ~2 folds in the DIO mice compared to the lean controls. We also observed significantly higher mRNA and serum levels of TNF-α in the DIO mice as compared to the lean mice. Higher TNF-α levels are known to be associated with liver toxicity. Conclusion: CPT-11 dosage should be closely monitored for effective and safe chemotherapy in obese patients who are at a higher risk of developing liver toxicity

Restricted Specificity of Xenopus TFIIIA for Transcription of Somatic 5S rRNA Genes

Xenopus transcription factor IIIA (TFIIIA) is phosphorylated on serine-16 by CK2. Replacements with alanine or glutamic acid were made at this position in order to address the question of whether phosphorylation possibly influences the function of this factor. Neither substitution has an effect on the DNA or RNA binding activity of TFIIIA. The wild-type factor and the alanine variant activate transcription of somatic- and oocyte-type 5S rRNA genes in nuclear extract immunodepleted of endogenous TFIIIA. The glutamic acid variant (S16E) supports the transcription of somatic-type genes at levels comparable to those of wild-type TFIIIA; however, there is no transcription of the oocyte-type genes. This differential behavior of the phosphomimetic mutant protein is also observed in vivo when using early-stage embryos, where this mutant failed to activate transcription of the endogenous oocyte-type genes. Template exclusion assays establish that the S16E mutant binds to the oocyte-type 5S rRNA genes and recruits at least one other polymerase III transcription factor into an inactive complex. Phosphorylation of TFIIIA by CK2 may allow the factor to continue to act as a positive activator of the somatic-type genes and simultaneously as a repressor of the oocyte-type 5S rRNA genes, indicating that there is a mechanism that actively promotes repression of the oocyte-type genes at the end of oogenesis

Role of Constitutive Androstane Receptor in Toll-Like Receptor-Mediated Regulation of Gene Expression of Hepatic Drug-Metabolizing Enzymes and Transporters

Impairment of drug disposition in the liver during inflammation has been attributed to downregulation of gene expression of drug-metabolizing enzymes (DMEs) and drug transporters. Inflammatory responses in the liver are primarily mediated by Toll-like receptors (TLRs). We have recently shown that activation of TLR2 or TLR4 by lipoteichoic acid (LTA) and lipopolysaccharide (LPS), respectively, leads to the downregulation of gene expression of DMEs/transporters. However, the molecular mechanism underlying this downregulation is not fully understood. The xenobiotic nuclear receptors, pregnane X receptor (PXR) and constitutive androstane receptor (CAR), regulate the expression of DMEs/transporter genes. Downregulation of DMEs/transporters by LTA or LPS was associated with reduced expression of PXR and CAR genes. To determine the role of CAR, we injected CAR(+/+) and CAR(-/-) mice with LTA or LPS, which significantly downregulated (~40%-60%) RNA levels of the DMEs, cytochrome P450 (Cyp)3a11, Cyp2a4, Cyp2b10, uridine diphosphate glucuronosyltransferase 1a1, amine N-sulfotransferase, and the transporter, multidrug resistance-associated protein 2, in CAR(+/+) mice. Suppression of most of these genes was attenuated in LTA-treated CAR(-/-) mice. In contrast, LPS-mediated downregulation of these genes was not attenuated in CAR(-/-) mice. Induction of these genes by mouse CAR activator 1,4-bis-[2-(3,5-dichloropyridyloxy)]benzene was sustained in LTA- but not in LPS-treated mice. Similar observations were obtained in humanized CAR mice. We have replicated these results in primary hepatocytes as well. Thus, LPS can downregulate DME/transporter genes in the absence of CAR, whereas the effect of LTA on these genes is attenuated in the absence of CAR, indicating the potential involvement of CAR in LTA-mediated downregulation of DME/transporter genes

Differential Role of Toll-Interleukin 1 Receptor Domain-Containing Adaptor Protein in Toll-Like Receptor 2-Mediated Regulation of Gene Expression of Hepatic Cytokines and Drug-Metabolizing EnzymesS⃞

Pharmacological activities of drugs are impaired during inflammation because of reduced expression of hepatic drug-metabolizing enzyme genes (DMEs) and their regulatory nuclear receptors (NRs): pregnane X receptor (PXR), constitutive androstane receptor (CAR), and retinoid X receptor (RXRα). We have shown that a component of Gram-positive bacteria, lipoteichoic acid (LTA) induces proinflammatory cytokines and reduces gene expression of hepatic DMEs and NRs. LTA is a Toll-like receptor 2 (TLR2) ligand, which initiates signaling by recruitment of Toll-interleukin 1 receptor domain-containing adaptor protein (TIRAP) to the cytoplasmic TIR domain of TLR2. To determine the role of TIRAP in TLR2-mediated regulation of DME genes, TLR2(+/+), TLR2(−/−), TIRAP(+/+), and TIRAP(−/−) mice were given LTA injections. RNA levels of the DMEs (Cyp3a11, Cyp2b10, and sulfoaminotransferase), xenobiotic NRs (PXR and CAR), and nuclear protein levels of the central NR RXRα were reduced ∼50 to 60% in LTA-treated TLR2(+/+) but not in TLR2(−/−) mice. Induction of hepatic cytokines (interleukin-1β, tumor necrosis factor-α, and interleukin-6), c-Jun NH2-terminal kinase, and nuclear factor-κΒ was blocked in TLR2(−/−) mice. As expected, expression of hepatic DMEs and NRs was reduced by LTA in TIRAP(+/+) but not in TIRAP(−/−) mice. Of interest, cytokine RNA levels were induced in the livers of both the TIRAP(+/+) and TIRAP(−/−) mice, whereas LTA-mediated induction of serum cytokines was attenuated in TIRAP(−/−) mice. LTA-mediated down-regulation of DME genes was attenuated in hepatocytes from TLR2(−/−) or TIRAP(−/−) mice and in small interfering RNA-treated hepatocytes. Thus, the effect of TLR2 on DME genes in hepatocytes was mediated by TIRAP, whereas TIRAP was not involved in mediating the effects of TLR2 on cytokine expression in the liver