Romi Ghose

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

BackgroundLipopolysaccharide (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.ResultsNuclear 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.ConclusionThe 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.

Nuclear Export of Retinoid X Receptor α in Response to Interleukin-1β-mediated Cell Signaling ROLES FOR JNK AND SER260

As the obligate heterodimer partner to class II nuclear receptors, the retinoid X receptor α (RXRα) plays a vital physiological role in the regulation of multiple hepatic functions, including bile formation, intermediary metabolism, and endobiotic/xenobiotic detoxification. Many RXRα-regulated genes are themselves suppressed in inflamed liver via unknown mechanisms, which constitute a substantial component of the negative hepatic acute phase response. In this study we show that RXRα, generally considered a stable nuclear resident protein, undergoes rapid nuclear export in response to signals initiated by the pro-inflammatory cytokine interleukin-1β (IL-1β), a central activator of the acute phase response. Within 30 min of exposure to IL-1β, nuclear levels of RXRα are markedly suppressed in human liver-derived HepG2 cells, temporally coinciding with its appearance in the cytoplasm. The nuclear residence of RXRα is maintained by inhibiting c-jun N-terminal kinase (JNK, curcumin or SP600125) or CRM-1-mediated nuclear export (Leptomycin B). Pretreatment with the proteasome inhibitor MG132 blocks IL-1β-mediated reductions in nuclear RXRα levels while increasing accumulation in the cytoplasm. Mutational studies identify one residue, serine 260, a JNK phosphoacceptor site whose phosphorylation status had an unknown role in RXRα function, as critical for IL-1β-mediated nuclear export of transfected human RXRα-green fluorescent fusion constructs. These findings indicate that inflammation-mediated cell signaling leads to rapid and profound reductions in nuclear RXRα levels, via a multistep, JNK-dependent mechanism involving Ser260, nuclear export, and proteasomal degradation. Thus, inflammation-meditated cell signaling targets RXRα for nuclear export and degradation; a potential mechanism that explains the broad suppression of RXRα-dependent gene expression in the inflamed liver.

Induction of TAK (Cyclin T1/P-TEFb) in Purified Resting CD4+ T Lymphocytes by Combination of Cytokines

ABSTRACT Combinations of cytokines are known to reactivate transcription and replication of latent human immunodeficiency virus type 1 (HIV-1) proviruses in resting CD4+ T lymphocytes isolated from infected individuals. Transcription of the HIV-1 provirus by RNA polymerase II is strongly stimulated by the viral Tat protein. Tat function is mediated by a cellular protein kinase known as TAK (cyclin T1/P-TEFb) that is composed of Cdk9 and cyclin T1. We have found that treatment of peripheral blood lymphocytes and purified resting CD4+ T lymphocytes with the combination of interleukin-2 (IL-2), IL-6, and tumor necrosis factor alpha resulted in an increase in Cdk9 and cyclin T1 protein levels and an increase in TAK enzymatic activity. The cytokine induction of TAK in resting CD4+ T lymphocytes did not appear to require proliferation of lymphocytes. These results suggest that induction of TAK by cytokines secreted in the microenvironment of lymphoid tissue may be involved in the reactivation of HIV-1 in CD4+ T lymphocytes harboring a latent provirus.

Role of high-fat diet in regulation of gene expression of drug metabolizing enzymes and transporters

AIM Our aim is to investigate the molecular mechanism of regulation of gene expression of drug metabolizing enzymes (DMEs) and transporters in diet-induced obesity. MAIN METHODS Adult male CD1 mice were fed diets containing 60% kcal fat (HFD) or 10% kcal fat (LFD) for 14 weeks. RNA levels of hepatic DMEs, transporters and their regulatory nuclear receptors (NRs) were analyzed by real-time PCR. Activation of cell-signaling components (JNK and NF-κΒ) and pro-inflammatory cytokines (IL-1β, IL-6 and TNFα) were measured in the liver. Finally, the pharmacodynamics of drugs metabolized by DMEs was measured to determine the clinical relevance of our findings. KEY FINDINGS RNA levels of the hepatic phase I (Cyp3a11, Cyp2b10, Cyp2a4) and phase II (Ugt1a1, Sult1a1, Sultn) enzymes were reduced ~30-60% in HFD compared to LFD mice. RNA levels of Cyp2e1, Cyp1a2 and the drug transporters, multidrug resistance proteins, (Mrp)2, Mrp3 and multidrug resistant gene (Mdr)1b were unaltered in HFD mice. Gene expression of the NRs, PXR and CAR and nuclear protein levels of RXRα was reduced in HFD mice. Cytokines, JNK and NF-κΒ were induced in HFD mice. Thus reduction in hepatic gene expression in obesity may be modulated by cross-talk between NRs and inflammation-induced cell-signaling. Sleep time of Midazolam (Cyp3a substrate) was prolonged in HFD mice, while Zoxazolamine (Cyp1a2 and Cyp2e1 substrate)-induced sleep time was unaltered. SIGNIFICANCE This study demonstrates that gene-specific reductions in DMEs can affect specific drugs metabolized by these enzymes, thus providing a rationale to monitor the effectiveness of drug therapy in obese individuals.

Antiapoptotic Function of Cdk9 (TAK/P-TEFb) in U937 Promonocytic Cells

ABSTRACT Cdk9 is the catalytic subunit of TAK (cyclinT1/P-TEFb), a cellular protein kinase that mediates human immunodeficiency virus type 1 (HIV-1) Tat transcriptional activation function. To examine Cdk9 function in cells relevant to HIV-1 infection, we used a murine leukemia virus retrovirus vector to transduce and overexpress the cDNA of a dominant negative mutant Cdk9 protein (Cdk9-dn) in Jurkat T cells and U937 promonocytic cells. In Jurkat cells, overexpression of Cdk9-dn specifically inhibited Tat transactivation and HIV-1 replication but had no inhibitory effect on induction of CD69, CD25, and interleukin-2 following T-cell activation. In U937 cells, overexpression of Cdk9-dn sensitized cells to apoptosis, especially after phorbol myristate acetate (PMA) treatment to induce differentiation to macrophage-like cells. Because Cdk9 function is induced in PMA-treated U937 cells, Cdk9 may play an antiapoptotic role during monocyte differentiation.

Drug disposition in pathophysiological conditions.

Expression and activity of several key drug metabolizing enzymes (DMEs) and transporters are altered in various pathophysiological conditions, leading to altered drug metabolism and disposition. This can have profound impact on the pharmacotherapy of widely used clinically relevant medications in terms of safety and efficacy by causing inter-individual variabilities in drug responses. This review article highlights altered drug disposition in inflammation and infectious diseases, and commonly encountered disorders such as cancer, obesity/diabetes, fatty liver diseases, cardiovascular diseases and rheumatoid arthritis. Many of the clinically relevant drugs have a narrow therapeutic index. Thus any changes in the disposition of these drugs may lead to reduced efficacy and increased toxicity. The implications of changes in DMEs and transporters on the pharmacokinetics/pharmacodynamics of clinically-relevant medications are also discussed. Inflammation-mediated release of pro-inflammatory cytokines and activation of toll-like receptors (TLRs) are known to play a major role in down-regulation of DMEs and transporters. Although the mechanism by which this occurs is unclear, several studies have shown that inflammation-associated cell-signaling pathway and its interaction with basal transcription factors and nuclear receptors in regulation of DMEs and transporters play a significant role in altered drug metabolism. Altered regulation of DMEs and transporters in a multitude of disease states will contribute towards future development of powerful in vitro and in vivo tools in predicting the drug response and opt for better drug design and development. The goal is to facilitate a better understanding of the mechanistic details underlying the regulation of DMEs and transporters in pathophysiological conditions.

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 Enzymes

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.

Regulation of gene expression of hepatic drug metabolizing enzymes and transporters by the Toll-like receptor 2 ligand, Lipoteichoic acid

Expression of hepatic drug metabolizing enzymes (DMEs) is altered in infection and inflammation. However, the role of Gram+ve bacterial components and their receptor, Toll-like receptor (TLR) 2 in regulation of hepatic DMEs is unknown. Gene expression of DMEs is regulated by members of the nuclear receptor superfamily (PXR, CAR and RXRalpha). The TLR2 ligand, lipoteichoic acid (LTA) reduced RNA levels of CAR and its target genes, Cyp2b10, Cyp2a4 and Sultn in mouse liver ( approximately 60-80% reduction). Hepatic genes regulated by PXR and CAR, Cyp3a11 and Mrp2 were moderately reduced by LTA, along with approximately 50% reduction of PXR RNA and nuclear protein levels of RXRalpha. The effects of LTA were significantly attenuated by pre-treatment with the Kupffer cell inhibitor, gadolinium chloride, indicating that Kupffer cells contribute to LTA-mediated down-regulation of hepatic genes. These results indicate that treatment with Gram+ve bacterial components preferentially down-regulate CAR and its target genes in the liver.

Regulation of Hepatic Drug-Metabolizing Enzyme Genes by Toll-Like Receptor 4 Signaling Is Independent of Toll-Interleukin 1 Receptor Domain-Containing Adaptor Protein

During inflammation, drug metabolism and clearance are altered due to suppression of hepatic drug-metabolizing enzyme (DME) genes and their regulatory nuclear receptors (NRs) [pregnane X receptor, constitutive androstane receptor, and retinoid X receptor α (RXRα)]. The bacterial endotoxin, lipopolysaccharide (LPS), induces expression of proinflammatory cytokines in the liver, which contribute to altered DME expression. LPS binds to the cell-surface receptor, Toll-like receptor 4 (TLR4), which initiates a signal transduction cascade, including recruitment of the Toll-interleukin 1 receptor domain-containing adaptor protein (TIRAP). However, the role of TLR4 and TIRAP in LPS-mediated regulation of hepatic DME gene expression is not known. Wild-type (C3HeB/FeJ), TLR4-mutant (C3H/HeJ), TIRAP+/+, and TIRAP-/- mice were injected i.p. with LPs. RNA levels of the major hepatic DME, Cyp3a11 and Ugt1a1, and the NRs were suppressed ∼60 to 70% by LPS in wild-type but not in the TLR4-mutant mice. The nuclear protein levels of RXRα were reduced by LPS in wild-type but not in TLR4-mutant mice. Induction of hepatic cytokines (interleukin-1β, tumor necrosis factor-α, and interleukin-6), c-Jun N-terminal kinase, and nuclear factor-κB was blocked in TLR4-mutant mice. Surprisingly, LPS had the same effect on cytokines, kinases, NRs, and DME genes in livers of both TIRAP+/+ and TIRAP-/- mice, indicating that TIRAP is not essential for TLR4-mediated suppression of NRs and DMEs in liver. However, TIRAP-/- mice have reduced serum cytokine expression compared with TIRAP+/+ mice in response to LPS. This shows that although TIRAP mediates inflammatory responses induced by LPS, it is not essential in regulating LPS-mediated alterations of gene expression in liver.

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.