[Show abstract][Hide abstract] ABSTRACT: Chronic kidney disease (CKD) is a major health issue for HIV-positive individuals, associated with increased morbidity and mortality. Development and implementation of a risk score model for CKD would allow comparison of the risks and benefits of adding potentially nephrotoxic antiretrovirals to a treatment regimen and would identify those at greatest risk of CKD. The aims of this study were to develop a simple, externally validated, and widely applicable long-term risk score model for CKD in HIV-positive individuals that can guide decision making in clinical practice.
[Show abstract][Hide abstract] ABSTRACT: The early region 1A (E1A) of human adenovirus types 2 and 5 are differentially spliced to yield five distinct mRNAs that encode different proteins. The smallest E1A RNA transcript encodes a 55 residue (R) protein that shares only 28 amino acid residues in common with the other E1A proteins. Even though it is the most abundant E1A transcript at late times post infection, little is known about the functions of this E1A isoform. In this study, we show that the E1A 55R protein interacts with, and modulates the activity of the unliganded thyroid hormone receptor (TR). We demonstrate that E1A 55R contains a signature motif known as the CoRNR box that confers interaction with the unliganded thyroid hormone receptor (TR) and was originally identified in cellular corepressors. Using a system reconstituted in the yeast Saccharomyces cerevisiae, which lack endogenous TR and TR coregulators, we show that E1A 55R nonetheless differs from cellular corepressors as it functions as a strong co-activator of TR dependent transcription and that it possesses an intrinsic transcriptional activation domain. These data indicate that the E1A 55R protein functions as a transcriptional regulator.
Full-text · Article · Oct 2013 · Journal of General Virology
[Show abstract][Hide abstract] ABSTRACT: Background
Non-nucleoside reverse transcriptase inhibitor (NNRTI)-resistant mutants have been shown to emerge after interruption of suppressive NNRTI-based antiretroviral therapy (ART) using routine testing. The aim of this study was to quantify the risk of resistance by sensitive testing and correlate the detection of resistance with NNRTI concentrations after treatment interruption and virologic responses after treatment resumption.
Resistance-associated mutations (RAMs) and NNRTI concentrations were studied in plasma from 132 patients who interrupted suppressive ART within SMART. RAMs were detected by Sanger sequencing, allele-specific PCR, and ultra-deep sequencing. NNRTI concentrations were measured by sensitive high-performance liquid chromatography.
Four weeks after NNRTI interruption, 19/31 (61.3%) and 34/39 (87.2%) patients showed measurable nevirapine (>0.25 ng/ml) or efavirenz (>5 ng/ml) concentrations, respectively. Median eight weeks after interruption, 22/131 (16.8%) patients showed ≥1 NNRTI-RAM, including eight patients with NNRTI-RAMs detected only by sensitive testing. The adjusted odds ratio (OR) of NNRTI-RAM detection was 7.62 (95% confidence interval [CI] 1.52, 38.30; p = 0.01) with nevirapine or efavirenz concentrations above vs. below the median measured in the study population. Staggered interruption, whereby nucleos(t)ide reverse transcriptase inhibitors (NRTIs) were continued for median nine days after NNRTI interruption, did not prevent NNRTI-RAMs, but increased detection of NRTI-RAMs (OR 4.25; 95% CI 1.02, 17.77; p = 0.03). After restarting NNRTI-based ART (n = 90), virologic suppression rates <400 copies/ml were 8/13 (61.5%) with NNRTI-RAMs, 7/11 (63.6%) with NRTI-RAMs only, and 51/59 (86.4%) without RAMs. The ORs of re-suppression were 0.18 (95% CI 0.03, 0.89) and 0.17 (95% CI 0.03, 1.15) for patients with NNRTI-RAMs or NRTI-RAMs only respectively vs. those without RAMs (p = 0.04).
Detection of resistant mutants in the rebound viremia after interruption of efavirenz- or nevirapine-based ART affects outcomes once these drugs are restarted. Further studies are needed to determine RAM persistence in untreated patients and impact on newer NNRTIs.
[Show abstract][Hide abstract] ABSTRACT: Liver specific thyroid hormone receptor β (TRβ) specific agonists are potent lipid lowering drugs that also hold promise for treating nonalcoholic fatty liver disease (NAFLD) and hepatic insulin resistance. We investigated the effect of two TRβ agonists (GC-1 and KB-2115) in high-fat fed male Sprague Dawley rats treated for 10 days. GC-1 treatment reduced hepatic triglyceride content by 75%, but the rats developed fasting hyperglycemia and hyperinsulinemia, attributable to increased endogenous glucose production (EGP) and diminished hepatic insulin sensitivity. GC-1 also increased white adipose tissue lipolysis; the resulting increase in glycerol flux may have contributed to the increase in EGP. KB-2115, a more TRβ and liver specific thyromimetic, also prevented hepatic steatosis, but did not induce fasting hyperglycemia, increase basal EGP rate or diminish hepatic insulin sensitivity. Surprisingly, insulin stimulated peripheral glucose disposal was diminished due to a decrease in insulin stimulated skeletal muscle glucose uptake. Skeletal muscle insulin signaling was unaffected. Instead, KB2115 treatment was associated with a decrease in GLUT4 protein content. Thus, though both GC-1 and KB-2115 potently treat hepatic steatosis in fat-fed rats, they each worsen insulin action via specific and discrete mechanisms. The development of future TRβ agonists must consider the potential adverse effects on insulin sensitivity.
No preview · Article · May 2013 · AJP Endocrinology and Metabolism
[Show abstract][Hide abstract] ABSTRACT: The majority of cholesterol reduction therapies, such as the statin drugs, work primarily by inducing the expression of hepatic low-density lipoprotein receptors (LDLRs), rendering these therapeutics only partially effective in animals lacking LDLRs. Although thyroid hormones and their synthetic derivatives, often referred to as thyromimetics, have been clearly shown to reduce serum cholesterol levels, this action has generally been attributed to their ability to increase expression of hepatic LDLRs. Here we show for the first time that the thyroid hormone T(3) and the thyroid hormone receptor-β selective agonists GC-1 and KB2115 are capable of markedly reducing serum cholesterol in mice devoid of functional LDLRs by inducing Cyp7a1 expression and stimulating the conversion and excretion of cholesterol as bile acids. Based on this LDLR-independent mechanism, thyromimetics such as GC-1 and KB2115 may represent promising cholesterol-lowering therapeutics for the treatment of diseases such as homozygous familial hypercholesterolemia, a rare genetic disorder caused by a complete lack of functional LDLRs, for which there are limited treatment options because most therapeutics are only minimally effective.
[Show abstract][Hide abstract] ABSTRACT: Thyroid hormone receptor (TR)/peroxisome proliferator activated receptor coactivator (PGC-1α) interactions are required for T(3)-dependent transcriptional responses involved in adaptive thermogenesis and liver. Thus, it is important to define TR/PGC-1α contact modes and to understand their significance in gene expression. Previous studies have shown that TRβ1 recruits PGC-1α to target promoters via contacts between the hormone-dependent TRβ1 activation function 2 (AF-2) in the C-terminal ligand binding domain (LBD) and a major PGC-1α nuclear receptor (NR) interaction box (consensus LxxLL) at amino acids 142-146. While our studies verify the existence and importance of this interaction, we present evidence that TRβ1 also binds PGC-1α in a second ligand and LxxLL motif independent mode and show that this interaction requires the TRβ1 N-terminal domain (NTD) and the PGC-1α N-terminal activation domain (AD) at amino acids 1-130. Transfection assays suggest that optimal PGC-1α coactivation requires the TRβ1 NTD and that these contacts are needed for utilization of the PGC-1α C-terminal AD, which does not bind TR and is implicated in basal transcription machinery contacts. We propose that TR AF-1/PGC-1α contacts are needed for transition between activities of PGC-1α N-and C-terminal ADs in gene expression. Our findings provide insights into possible roles for TR and NR AF-1 in gene expression.
No preview · Article · Sep 2012 · The Journal of steroid biochemistry and molecular biology
[Show abstract][Hide abstract] ABSTRACT: Androgen receptor (AR) is a major therapeutic target that plays pivotal roles in prostate cancer (PCa) and androgen insensitivity syndromes. We previously proposed that compounds recruited to ligand-binding domain (LBD) surfaces could regulate AR activity in hormone-refractory PCa and discovered several surface modulators of AR function. Surprisingly, the most effective compounds bound preferentially to a surface of unknown function [binding function 3 (BF-3)] instead of the coactivator-binding site [activation function 2 (AF-2)]. Different BF-3 mutations have been identified in PCa or androgen insensitivity syndrome patients, and they can strongly affect AR activity. Further, comparison of AR x-ray structures with and without bound ligands at BF-3 and AF-2 showed structural coupling between both pockets. Here, we combine experimental evidence and molecular dynamic simulations to investigate whether BF-3 mutations affect AR LBD function and dynamics possibly via allosteric conversation between surface sites. Our data indicate that AF-2 conformation is indeed closely coupled to BF-3 and provide mechanistic proof of their structural interconnection. BF-3 mutations may function as allosteric elicitors, probably shifting the AR LBD conformational ensemble toward conformations that alter AF-2 propensity to reorganize into subpockets that accommodate N-terminal domain and coactivator peptides. The induced conformation may result in either increased or decreased AR activity. Activating BF-3 mutations also favor the formation of another pocket (BF-4) in the vicinity of AF-2 and BF-3, which we also previously identified as a hot spot for a small compound. We discuss the possibility that BF-3 may be a protein-docking site that binds to the N-terminal domain and corepressors. AR surface sites are attractive pharmacological targets to develop allosteric modulators that might be alternative lead compounds for drug design.
Full-text · Article · May 2012 · Molecular Endocrinology
[Show abstract][Hide abstract] ABSTRACT: The recent discovery that peroxisome proliferator-activated receptor γ (PPARγ) targeted anti-diabetic drugs function by inhibiting Cdk5-mediated phosphorylation of the receptor has provided a new viewpoint to evaluate and perhaps develop improved insulin-sensitizing agents. Herein we report the development of a novel thiazolidinedione that retains similar anti-diabetic efficacy as rosiglitazone in mice yet does not elicit weight gain or edema, common side effects associated with full PPARγ activation. Further characterization of this compound shows GQ-16 to be an effective inhibitor of Cdk5-mediated phosphorylation of PPARγ. The structure of GQ-16 bound to PPARγ demonstrates that the compound utilizes a binding mode distinct from other reported PPARγ ligands, although it does share some structural features with other partial agonists, such as MRL-24 and PA-082, that have similarly been reported to dissociate insulin sensitization from weight gain. Hydrogen/deuterium exchange studies reveal that GQ-16 strongly stabilizes the β-sheet region of the receptor, presumably explaining the compound's efficacy in inhibiting Cdk5-mediated phosphorylation of Ser-273. Molecular dynamics simulations suggest that the partial agonist activity of GQ-16 results from the compound's weak ability to stabilize helix 12 in its active conformation. Our results suggest that the emerging model, whereby "ideal" PPARγ-based therapeutics stabilize the β-sheet/Ser-273 region and inhibit Cdk5-mediated phosphorylation while minimally invoking adipogenesis and classical agonism, is indeed a valid framework to develop improved PPARγ modulators that retain antidiabetic actions while minimizing untoward effects.
Full-text · Article · May 2012 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: Gene expression is tightly regulated by transcription factors and cofactors that function by directly or indirectly interacting with DNA of the genome. Understanding how and where these proteins bind provides essential information to uncover genetic regulatory mechanisms. We have developed a new method to study DNA-protein interaction in vivo called DNA adenine methyltransferase (Dam)IP, which is based on fusing a protein of interest to a mutant form of Dam from Escherichia coli. We showed previously that DamIP can efficiently identify in vivo binding sites of Dam-tethered human estrogen receptor (hER)α. In current study, we present the cistrome of hERα determined by DamIP and high throughput sequencing (DamIP-seq). The DamIP-seq-defined hERα cistrome identifies many new binding regions and overlaps with those determined by chromatin immunoprecipitation (ChIP)-chip or ChIP-seq. Elements uniquely identified by DamIP-seq include a unique class of elements that show low, but persistent, hERα binding when reexamined by conventional ChIP. In contrast, DamIP-seq fails to detect some elements with very transient hERα binding. The methyl-adenine modifications introduced by Dam are stable and do not decrease over 12 d. In summary, the current study provides both an alternate view of the hERα cistrome to further understand the mechanism of hERα-mediated transcription and a new tool to explore other transcriptional factors and cofactors that is very different from conventional ChIP.
No preview · Article · Dec 2011 · Molecular Endocrinology
[Show abstract][Hide abstract] ABSTRACT: We report the three-dimensional structure of a β-catenin armadillo repeat in complex with the liver receptor homolog-1 (LRH-1) ligand binding domain at 2.8 Å resolution as the first structure of β-catenin in complex with any nuclear receptor. The surface of β-catenin that binds LRH-1 partly overlaps defined contact sites for peptide segments of β-catenin partners, including T-cell factor-4. The surface of LRH-1 that engages β-catenin is comprised of helices 1, 9, and 10 and is distinct from known interaction surfaces of LRH-1, including corepressor and coactivator binding sites. Targeted mutagenesis of amino acids forming both sides of the LRH-1/β-catenin interface reveals that they are essential for stable interactions between these proteins in solution. The LRH-1 binding site in β-catenin is also required for association with androgen receptor, providing evidence that the observed LRH-1/β-catenin interaction may be prototypic.
Preview · Article · Dec 2011 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: RG and RP do not alter feeding intake. Food consumption was measured per cage during the 7 weeks of treatment. Normalized daily food intake in mg food/g body weight/day is given for each week of treatment.
[Show abstract][Hide abstract] ABSTRACT: Long-term estrogen deficiency increases the risk of obesity, diabetes and metabolic syndrome in postmenopausal women. Menopausal hormone therapy containing estrogens might prevent these conditions, but its prolonged use increases the risk of breast cancer, as wells as endometrial cancer if used without progestins. Animal studies indicate that beneficial effects of estrogens in adipose tissue and adverse effects on mammary gland and uterus are mediated by estrogen receptor alpha (ERα). One strategy to improve the safety of estrogens to prevent/treat obesity, diabetes and metabolic syndrome is to develop estrogens that act as agonists in adipose tissue, but not in mammary gland and uterus. We considered plant extracts, which have been the source of many pharmaceuticals, as a source of tissue selective estrogens. Extracts from two plants, Glycyrrhiza uralensis (RG) and Pueraria montana var. lobata (RP) bound to ERα, activated ERα responsive reporters, and reversed weight gain and fat accumulation comparable to estradiol in ovariectomized obese mice maintained on a high fat diet. Unlike estradiol, RG and RP did not induce proliferative effects on mammary gland and uterus. Gene expression profiling demonstrated that RG and RP induced estradiol-like regulation of genes in abdominal fat, but not in mammary gland and uterus. The compounds in extracts from RG and RP might constitute a new class of tissue selective estrogens to reverse weight gain, fat accumulation and metabolic syndrome in postmenopausal women.
[Show abstract][Hide abstract] ABSTRACT: Synthetic selective thyroid hormone (TH) receptor (TR) modulators (STRM) exhibit beneficial effects on dyslipidemias in animals and humans and reduce obesity, fatty liver, and insulin resistance in preclinical animal models. STRM differ from native TH in preferential binding to the TRβ subtype vs. TRα, increased uptake into liver, and reduced uptake into other tissues. However, selective modulators of other nuclear receptors exhibit important gene-selective actions, which are attributed to differential effects on receptor conformation and dynamics and can have profound influences in animals and humans. Although there are suggestions that STRM may exhibit such gene-specific actions, the extent to which they are actually observed in vivo has not been explored. Here, we show that saturating concentrations of the main active form of TH, T(3), and the prototype STRM GC-1 induce identical gene sets in livers of euthyroid and hypothyroid mice and a human cultured hepatoma cell line that only expresses TRβ, HepG2. We find one case in which GC-1 exhibits a modest gene-specific reduction in potency vs. T(3), at angiopoietin-like factor 4 in HepG2. Investigation of the latter effect confirms that GC-1 acts through TRβ to directly induce this gene but this gene-selective activity is not related to unusual T(3)-response element sequence, unlike previously documented promoter-selective STRM actions. Our data suggest that T(3) and GC-1 exhibit almost identical gene regulation properties and that gene-selective actions of GC-1 and similar STRM will be subtle and rare.
[Show abstract][Hide abstract] ABSTRACT: Peroxisome proliferator-activated receptor γ (PPARγ) activation induces adipogenesis and also enhances lipogenesis, mitochondrial
activity, and insulin sensitivity in adipocytes. Whereas some studies implicate PPARγ coactivator 1α (PGC-1α) in the mitochondrial
effect, the mechanisms involved in PPARγ regulation of adipocyte mitochondrial function are not resolved. PPARγ-activating
ligands (thiazolidinediones (TZDs)) are important insulin sensitizers and were recently shown to indirectly induce PGC-1β
transcription in osteoclasts. Here, we asked whether similar effects occur in adipocytes and show that TZDs also strongly
induce PGC-1β in cultured 3T3-L1 cells. This effect, however, differs from the indirect effect proposed for bone and is rapid
and direct and involves PPARγ interactions with an intronic PPARγ response element cluster in the PGC-1β locus. TZD treatment
of cultured adipocytes results in up-regulation of mitochondrial marker genes, and increased mitochondrial activity and use
of short interfering RNA confirms that these effects require PGC-1β. PGC-1β did not participate in PPARγ effects on adipogenesis
or lipogenesis, and PGC-1β knockdown did not alter insulin-responsive glucose uptake into 3T3-L1 cells. Similar effects on
PGC-1β and mitochondrial gene expression are seen in vivo; fractionation of obese mouse adipose tissue reveals that PPARγ and PGC-1β, but not PGC-1α, are coordinately up-regulated
in adipocytes relative to preadipocytes and that TZD treatment induces PGC-1β and mitochondrial marker genes in adipose tissue
of obese mice. We propose that PPARγ directly induces PGC-1β expression in adipocytes and that this effect regulates adipocyte
No preview · Article · Sep 2011 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: Peroxisome proliferator-activated receptor γ (PPARγ) activation induces adipogenesis and also enhances lipogenesis, mitochondrial activity, and insulin sensitivity in adipocytes. Whereas some studies implicate PPARγ coactivator 1α (PGC-1α) in the mitochondrial effect, the mechanisms involved in PPARγ regulation of adipocyte mitochondrial function are not resolved. PPARγ-activating ligands (thiazolidinediones (TZDs)) are important insulin sensitizers and were recently shown to indirectly induce PGC-1β transcription in osteoclasts. Here, we asked whether similar effects occur in adipocytes and show that TZDs also strongly induce PGC-1β in cultured 3T3-L1 cells. This effect, however, differs from the indirect effect proposed for bone and is rapid and direct and involves PPARγ interactions with an intronic PPARγ response element cluster in the PGC-1β locus. TZD treatment of cultured adipocytes results in up-regulation of mitochondrial marker genes, and increased mitochondrial activity and use of short interfering RNA confirms that these effects require PGC-1β. PGC-1β did not participate in PPARγ effects on adipogenesis or lipogenesis, and PGC-1β knockdown did not alter insulin-responsive glucose uptake into 3T3-L1 cells. Similar effects on PGC-1β and mitochondrial gene expression are seen in vivo; fractionation of obese mouse adipose tissue reveals that PPARγ and PGC-1β, but not PGC-1α, are coordinately up-regulated in adipocytes relative to preadipocytes and that TZD treatment induces PGC-1β and mitochondrial marker genes in adipose tissue of obese mice. We propose that PPARγ directly induces PGC-1β expression in adipocytes and that this effect regulates adipocyte mitochondrial activity.
No preview · Article · Jun 2011 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND AND OBJECTIVES:
Bacterial pneumonia still contributes to morbidity/mortality in HIV infection despite effective combination antiretroviral therapy (cART). Evaluation of Subcutaneous Interleukin-2 in a Randomized International Trial (ESPRIT), a trial of intermittent recombinant interleukin-2 (rIL-2) with cART vs. cART alone (control arm) in HIV-infected adults with CD4 counts ≥300cells/μL, offered the opportunity to explore associations between bacterial pneumonia and rIL-2, a cytokine that increases the risk of some bacterial infections.
Baseline and time-updated factors associated with first-episode pneumonia on study were analysed using multivariate proportional hazards regression models. Information on smoking/pneumococcal vaccination history was not collected.
IL-2 cycling was most intense in years 1-2. Over ≈7 years, 93 IL-2 [rate 0.67/100 person-years (PY)] and 86 control (rate 0.63/100 PY) patients experienced a pneumonia event [hazard ratio (HR) 1.06; 95% confidence interval (CI) 0.79, 1.42; P=0.68]. Median CD4 counts prior to pneumonia were 570cells/μL (IL-2 arm) and 463cells/μL (control arm). Baseline risks for bacterial pneumonia included older age, injecting drug use, detectable HIV viral load (VL) and previous recurrent pneumonia; Asian ethnicity was associated with decreased risk. Higher proximal VL (HR for 1 log(10) higher VL 1.28; 95% CI 1.11, 1.47; P<0.001) was associated with increased risk; higher CD4 count prior to the event (HR per 100 cells/μL higher 0.94; 95% CI 0.89, 1.0; P=0.04) decreased risk. Compared with controls, the hazard for a pneumonia event was higher if rIL-2 was received <180 days previously (HR 1.66; 95% CI 1.07, 2.60; P=0.02) vs.≥180 days previously (HR 0.98; 95% CI 0.70, 1.37; P=0.9). Compared with the control group, pneumonia risk in the IL-2 arm decreased over time, with HRs of 1.41, 1.71, 1.16, 0.62 and 0.84 in years 1, 2, 3-4, 5-6 and 7, respectively.
Bacterial pneumonia rates in cART-treated adults with moderate immunodeficiency are high. The mechanism of the association between bacterial pneumonia and recent IL-2 receipt and/or detectable HIV viraemia warrants further exploration.