Nicotinamide Antagonizes Lipopolysaccharide-Induced Hypoxic Cell Signals in Human Macrophages.

Mechanisms to control the immune response are important to pathogen evasion and host defense. Gram-negative bacteria are common pathogens that can activate host immune responses through their outer membrane component, LPS. Macrophage activation by LPS induces cell signals that promote hypoxic metabolism, phagocytosis, Ag presentation, and inflammation. Nicotinamide (NAM) is a vitamin B3 derivative and precursor in the formation of NAD, which is a required cofactor in cellular function. In this study, treatment of human monocyte-derived macrophages with NAM promoted posttranslational modifications that antagonized LPS-induced cell signals. Specifically, NAM inhibited AKT and FOXO1 phosphorylation, decreased p65/RelA acetylation, and promoted p65/RelA and hypoxia-inducible transcription factor-1α (HIF-1α) ubiquitination. NAM also increased prolyl hydroxylase domain 2 (PHD2) production, inhibited HIF-1α transcription, and promoted the formation of the proteasome, resulting in reduced HIF-1α stabilization, decreased glycolysis and phagocytosis, and reductions in NOX2 activity and the production of lactate dehydrogenase A. These NAM responses were associated with increased intracellular NAD levels formed through the salvage pathway. NAM and its metabolites may therefore decrease the inflammatory response of macrophages and protect the host against excessive inflammation but potentially increase injury through reduced pathogen clearance. Continued study of NAM cell signals in vitro and in vivo may provide insight into infection-associated host pathologies and interventions.

Type I interferon activation and endothelial dysfunction in caveolin-1 insufficiency-associated pulmonary arterial hypertension.

Interferonopathies, interferon (IFN)-α/ß therapy, and caveolin-1 (CAV1) loss-of-function have all been associated with pulmonary arterial hypertension (PAH). Here, CAV1-silenced primary human pulmonary artery endothelial cells (PAECs) were proliferative and hypermigratory, with reduced cytoskeletal stress fibers. Signal transducers and activators of transcription (STAT) and phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) were both constitutively activated in these cells, resulting in a type I IFN-biased inflammatory signature. Cav1-/- mice that spontaneously develop pulmonary hypertension were found to have STAT1 and AKT activation in lung homogenates and increased circulating levels of CXCL10, a hallmark of IFN-mediated inflammation. PAH patients with CAV1 mutations also had elevated serum CXCL10 levels and their fibroblasts mirrored phenotypic and molecular features of CAV1-deficient PAECs. Moreover, immunofluorescence staining revealed endothelial CAV1 loss and STAT1 activation in the pulmonary arterioles of patients with idiopathic PAH, suggesting that this paradigm might not be limited to rare CAV1 frameshift mutations. While blocking JAK/STAT or AKT rescued aspects of CAV1 loss, only AKT inhibitors suppressed activation of both signaling pathways simultaneously. Silencing endothelial nitric oxide synthase (NOS3) prevented STAT1 and AKT activation induced by CAV1 loss, implicating CAV1/NOS3 uncoupling and NOS3 dysregulation in the inflammatory phenotype. Exogenous IFN reduced CAV1 expression, activated STAT1 and AKT, and altered the cytoskeleton of PAECs, implicating these mechanisms in PAH associated with autoimmune and autoinflammatory diseases, as well as IFN therapy. CAV1 insufficiency elicits an IFN inflammatory response that results in a dysfunctional endothelial cell phenotype and targeting this pathway may reduce pathologic vascular remodeling in PAH.

BMPR2 Loss Activates AKT by Disrupting DLL4/NOTCH1 and PPARγ Signaling in Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is a progressive cardiopulmonary disease characterized by pathologic vascular remodeling of small pulmonary arteries. Endothelial dysfunction in advanced PAH is associated with proliferation, apoptosis resistance, and endothelial to mesenchymal transition (EndoMT) due to aberrant signaling. DLL4, a cell membrane associated NOTCH ligand, plays a pivotal role maintaining vascular integrity. Inhibition of DLL4 has been associated with the development of pulmonary hypertension, but the mechanism is incompletely understood. Here we report that BMPR2 silencing in pulmonary artery endothelial cells (PAECs) activated AKT and suppressed the expression of DLL4. Consistent with these in vitro findings, increased AKT activation and reduced DLL4 expression was found in the small pulmonary arteries of patients with PAH. Increased NOTCH1 activation through exogenous DLL4 blocked AKT activation, decreased proliferation and reversed EndoMT. Exogenous and overexpression of DLL4 induced BMPR2 and PPRE promoter activity, and BMPR2 and PPARG mRNA in idiopathic PAH (IPAH) ECs. PPARγ, a nuclear receptor associated with EC homeostasis, suppressed by BMPR2 loss was induced and activated by DLL4/NOTCH1 signaling in both BMPR2-silenced and IPAH ECs, reversing aberrant phenotypic changes, in part through AKT inhibition. Directly blocking AKT or restoring DLL4/NOTCH1/PPARγ signaling may be beneficial in preventing or reversing the pathologic vascular remodeling of PAH.

Inflammation and DKK1-induced AKT activation contribute to endothelial dysfunction following NR2F2 loss.

NR2F2 is expressed in endothelial cells (ECs) and Nr2f2 knockout produces lethal cardiovascular defects. In humans, reduced NR2F2 expression is associated with cardiovascular diseases including congenital heart disease and atherosclerosis. Here, NR2F2 silencing in human primary ECs led to inflammation, endothelial-to-mesenchymal transition (EndMT), proliferation, hypermigration, apoptosis-resistance, and increased production of reactive oxygen species. These changes were associated with STAT and AKT activation along with increased production of DKK1. Co-silencing DKK1 and NR2F2 prevented NR2F2-loss-induced STAT and AKT activation and reversed EndMT. Serum DKK1 concentrations were elevated in patients with pulmonary arterial hypertension (PAH) and DKK1 was secreted by ECs in response to in vitro loss of either BMPR2 or CAV1, which are genetic defects associated with the development of PAH. In human primary ECs, NR2F2 suppressed DKK1, whereas its loss conversely induced DKK1 and disrupted endothelial homeostasis, promoting phenotypic abnormalities associated with pathologic vascular remodeling. Activating NR2F2 or blocking DKK1 may be useful therapeutic targets for treating chronic vascular diseases associated with EC dysfunction.NEW & NOTEWORTHY NR2F2 loss in the endothelial lining of blood vessels is associated with cardiovascular disease. Here, NR2F2-silenced human endothelial cells were inflammatory, proliferative, hypermigratory, and apoptosis-resistant with increased oxidant stress and endothelial-to-mesenchymal transition. DKK1 was induced in NR2F2-silenced endothelial cells, while co-silencing NR2F2 and DKK1 prevented NR2F2-loss-associated abnormalities in endothelial signaling and phenotype. Activating NR2F2 or blocking DKK1 may be useful therapeutic targets for treating vascular diseases associated with endothelial dysfunction.

Mineralocorticoid receptor antagonist treatment of established pulmonary arterial hypertension improves interventricular dependence in the SU5416-hypoxia rat model.

Treatment with mineralocorticoid receptor (MR) antagonists beginning at the outset of disease, or early thereafter, prevents pulmonary vascular remodeling in preclinical models of pulmonary arterial hypertension (PAH). However, the efficacy of MR blockade in established disease, a more clinically relevant condition, remains unknown. Therefore, we investigated the effectiveness of two MR antagonists, eplerenone (EPL) and spironolactone (SPL), after the development of severe right ventricular (RV) dysfunction in the rat SU5416-hypoxia (SuHx) PAH model. Cardiac magnetic resonance imaging (MRI) in SuHx rats at the end of week 5, before study treatment, confirmed features of established disease including reduced RV ejection fraction and RV hypertrophy, pronounced septal flattening with impaired left ventricular filling and reduced cardiac index. Five weeks of treatment with either EPL or SPL improved left ventricular filling and prevented the further decline in cardiac index compared with placebo. Interventricular septal displacement was reduced by EPL whereas SPL effects were similar, but not significant. Although MR antagonists did not significantly reduce pulmonary artery pressure or vessel remodeling in SuHx rats with established disease, animals with higher drug levels had lower pulmonary pressures. Consistent with effects on cardiac function, EPL treatment tended to suppress MR and proinflammatory gene induction in the RV. In conclusion, MR antagonist treatment led to modest, but consistent beneficial effects on interventricular dependence after the onset of significant RV dysfunction in the SuHx PAH model. These results suggest that measures of RV structure and/or function may be useful endpoints in clinical trials of MR antagonists in patients with PAH.

Mineralocorticoid Receptor (MR) trans-Activation of Inflammatory AP-1 Signaling: DEPENDENCE ON DNA SEQUENCE, MR CONFORMATION, AND AP-1 FAMILY MEMBER EXPRESSION.

Glucocorticoids are commonly used to treat inflammatory disorders. The glucocorticoid receptor (GR) can tether to inflammatory transcription factor complexes, such as NFκB and AP-1, and trans-repress the transcription of cytokines, chemokines, and adhesion molecules. In contrast, aldosterone and the mineralocorticoid receptor (MR) primarily promote cardiovascular inflammation by incompletely understood mechanisms. Although MR has been shown to weakly repress NFκB, its role in modulating AP-1 has not been established. Here, the effects of GR and MR on NFκB and AP-1 signaling were directly compared using a variety of ligands, two different AP-1 consensus sequences, GR and MR DNA-binding domain mutants, and siRNA knockdown or overexpression of core AP-1 family members. Both GR and MR repressed an NFκB reporter without influencing p65 or p50 binding to DNA. Likewise, neither GR nor MR affected AP-1 binding, but repression or activation of AP-1 reporters occurred in a ligand-, AP-1 consensus sequence-, and AP-1 family member-specific manner. Notably, aldosterone interactions with both GR and MR demonstrated a potential to activate AP-1. DNA-binding domain mutations that eliminated the ability of GR and MR to cis-activate a hormone response element-driven reporter variably affected the strength and polarity of these responses. Importantly, MR modulation of NFκB and AP-1 signaling was consistent with a trans-mechanism, and AP-1 effects were confirmed for specific gene targets in primary human cells. Steroid nuclear receptor trans-effects on inflammatory signaling are context-dependent and influenced by nuclear receptor conformation, DNA sequence, and the expression of heterologous binding partners. Aldosterone activation of AP-1 may contribute to its proinflammatory effects in the vasculature.

G Protein-coupled Receptor 40 (GPR40) and Peroxisome Proliferator-activated Receptor γ (PPARγ): AN INTEGRATED TWO-RECEPTOR SIGNALING PATHWAY.

Peroxisome proliferator-activated receptor γ (PPARγ) ligands have been widely used to treat type 2 diabetes mellitus. However, knowledge of PPARγ signaling remains incomplete. In addition to PPARγ, these drugs also activate G protein-coupled receptor 40 (GPR40), a Gαq-coupled free fatty acid receptor linked to MAPK networks and glucose homeostasis. Notably, p38 MAPK activation has been implicated in PPARγ signaling. Here, rosiglitazone (RGZ) activation of GPR40 and p38 MAPK was found to boost PPARγ-induced gene transcription in human endothelium. Inhibition or knockdown of p38 MAPK or expression of a dominant negative (DN) p38 MAPK mutant blunted RGZ-induced PPARγ DNA binding and reporter activity in EA.hy926 human endothelial cells. GPR40 inhibition or knockdown, or expression of a DN-Gαq mutant likewise blocked activation of both p38 MAPK and PPARγ reporters. Importantly, RGZ induction of PPARγ target genes in primary human pulmonary artery endothelial cells (PAECs) was suppressed by knockdown of either p38 MAPK or GPR40. GPR40/PPARγ signal transduction was dependent on p38 MAPK activation and induction of PPARγ co-activator-1 (PGC1α). Silencing of p38 MAPK or GPR40 abolished the ability of RGZ to induce phosphorylation and expression of PGC1α in PAECs. Knockdown of PGC1α, its essential activator SIRT1, or its binding partner/co-activator EP300 inhibited RGZ induction of PPARγ-regulated genes in PAECs. RGZ/GPR40/p38 MAPK signaling also led to EP300 phosphorylation, an event that enhances PPARγ target gene transcription. Thus, GPR40 and PPARγ can function as an integrated two-receptor signal transduction pathway, a finding with implications for rational drug development.

PPARγ signaling and emerging opportunities for improved therapeutics.

Peroxisome proliferator-activated receptor gamma (PPARγ) is a ligand-activated nuclear receptor that regulates glucose and lipid metabolism, endothelial function and inflammation. Rosiglitazone (RGZ) and other thiazolidinedione (TZD) synthetic ligands of PPARγ are insulin sensitizers that have been used for the treatment of type 2 diabetes. However, undesirable side effects including weight gain, fluid retention, bone loss, congestive heart failure, and a possible increased risk of myocardial infarction and bladder cancer, have limited the use of TZDs. Therefore, there is a need to better understand PPARγ signaling and to develop safer and more effective PPARγ-directed therapeutics. In addition to PPARγ itself, many PPARγ ligands including TZDs bind to and activate G protein-coupled receptor 40 (GPR40), also known as free fatty acid receptor 1. GPR40 signaling activates stress kinase pathways that ultimately regulate downstream PPARγ responses. Recent studies in human endothelial cells have demonstrated that RGZ activation of GPR40 is essential to the optimal propagation of PPARγ genomic signaling. RGZ/GPR40/p38 MAPK signaling induces and activates PPARγ co-activator-1α, and recruits E1A binding protein p300 to the promoters of target genes, markedly enhancing PPARγ-dependent transcription. Therefore in endothelium, GPR40 and PPARγ function as an integrated signaling pathway. However, GPR40 can also activate ERK1/2, a proinflammatory kinase that directly phosphorylates and inactivates PPARγ. Thus the role of GPR40 in PPARγ signaling may have important implications for drug development. Ligands that strongly activate PPARγ, but do not bind to or activate GPR40 may be safer than currently approved PPARγ agonists. Alternatively, biased GPR40 agonists might be sought that activate both p38 MAPK and PPARγ, but not ERK1/2, avoiding its harmful effects on PPARγ signaling, insulin resistance and inflammation. Such next generation drugs might be useful in treating not only type 2 diabetes, but also diverse chronic and acute forms of vascular inflammation such as atherosclerosis and septic shock.

Disruption of Ttll5/stamp gene (tubulin tyrosine ligase-like protein 5/SRC-1 and TIF2-associated modulatory protein gene) in male mice causes sperm malformation and infertility.

TTLL5/STAMP (tubulin tyrosine ligase-like family member 5) has multiple activities in cells. TTLL5 is one of 13 TTLLs, has polyglutamylation activity, augments the activity of p160 coactivators (SRC-1 and TIF2) in glucocorticoid receptor-regulated gene induction and repression, and displays steroid-independent growth activity with several cell types. To examine TTLL5/STAMP functions in whole animals, mice were prepared with an internal deletion that eliminated several activities of the Stamp gene. This mutation causes both reduced levels of STAMP mRNA and C-terminal truncation of STAMP protein. Homozygous targeted mutant (Stamp(tm/tm)) mice appear normal except for marked decreases in male fertility associated with defects in progressive sperm motility. Abnormal axonemal structures with loss of tubulin doublets occur in most Stamp(tm/tm) sperm tails in conjunction with substantial reduction in α-tubulin polyglutamylation, which closely correlates with the reduction in mutant STAMP mRNA. The axonemes in other structures appear unaffected. There is no obvious change in the organs for sperm development of WT versus Stamp(tm/tm) males despite the levels of WT STAMP mRNA in testes being 20-fold higher than in any other organ examined. This defect in male fertility is unrelated to other Ttll genes or 24 genes previously identified as important for sperm function. Thus, STAMP appears to participate in a unique, tissue-selective TTLL-mediated pathway for α-tubulin polyglutamylation that is required for sperm maturation and motility and may be relevant for male fertility.

Disruption of DLL4/NOTCH1 Causes Dysregulated PPARγ/AKT Signaling in Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is a progressive cardiopulmonary disease characterized by vascular remodeling of small pulmonary arteries. Endothelial dysfunction in advanced PAH is associated with proliferation, apoptosis resistance, and endothelial to mesenchymal transition (EndoMT) due to aberrant signaling. DLL4, a cell membrane associated NOTCH ligand, activates NOTCH1 signaling and plays a pivotal role maintaining vascular integrity. Inhibition of DLL4 has been associated with the development of pulmonary hypertension, but the mechanism is incompletely understood. Here we report that BMPR2 silencing in PAECs activated AKT and decreased DLL4 expression. DLL4 loss was also seen in lungs of patients with IPAH and HPAH. Over-expression of DLL4 in PAECs induced BMPR2 promoter activity and exogenous DLL4 increased BMPR2 mRNA through NOTCH1 activation. Furthermore, DLL4/NOTCH1 signaling blocked AKT activation, decreased proliferation and reversed EndoMT in BMPR2 - silenced PAECs and ECs from IPAH patients. PPARγ, suppressed by BMPR2 loss, was induced and activated by DLL4/NOTCH1 signaling in both BMPR2 -silenced and IPAH PAECs, reversing aberrant phenotypic changes, in part through AKT inhibition. Finally, leniolisib, a well-tolerated oral PI3K8/AKT inhibitor, decreased cell proliferation, induced apoptosis and reversed markers of EndoMT in BMPR2 -silenced PAECs. Restoring DLL4/NOTCH1/PPARγ signaling and/or suppressing AKT activation may be beneficial in preventing or reversing the pathologic vascular remodeling of PAH.

Identification of location and kinetically defined mechanism of cofactors and reporter genes in the cascade of steroid-regulated transactivation.

A currently obscure area of steroid hormone action is where the component factors, including receptor and reporter gene, act. The DNA binding of factors can be precisely defined, but the location and timing of factor binding and action are usually not equivalent. These questions are addressed for several factors (e.g. glucocorticoid receptor (GR), reporter, TIF2, NCoR, NELF-A, sSMRT, and STAMP) using our recently developed competition assay. This assay reveals both the kinetically defined mechanism of factor action and where the above factors act relative to both each other and the equilibrium equivalent to the rate-limiting step, which we call the concentration limiting step (CLS). The utility of this competition assay would be greatly increased if the position of the CLS is invariant and if the factor acting at the CLS is known. Here we report that the exogenous GREtkLUC reporter acts at the CLS as an accelerator for gene induction by GRs in U2OS cells. This mechanism of reporter function at the CLS persists with different reporters, factors, receptors, and cell types. We, therefore, propose that the reporter gene always acts at the CLS during gene induction and constitutes a landmark around which one can order the actions of all other factors. Current data suggest that how and where GR and the short form of SMRT act is also constant. These results validate a novel and rational methodology for identifying distally acting factors that would be attractive targets for pharmaceutical intervention in the treatment of diseases involving GR-regulated genes.

An evidence-based model comparing the cost-effectiveness of platelet-rich plasma gel to alternative therapies for patients with nonhealing diabetic foot ulcers.

OBJECTIVE: A cost-effectiveness analysis compared the potential economic benefit of an autologous, platelet-rich plasma (PRP) gel to alternative therapies in treating nonhealing diabetic foot ulcers. DESIGN: An economic model used peer-reviewed data to simulate clinical and cost outcomes and quality-adjusted life-years (QALYs) associated with PRP gel and other treatment modalities. PATIENTS: The model varies rates of healing, recurrence, infection, amputation, death, and associated costs for a hypothetical group of 200,000 patients with full-thickness, nonhealing diabetic foot ulcers for 5 years or until death. MAIN OUTCOME MEASURES: The model simulates the clinical, cost, and QALY outcomes associated with PRP gel versus other modalities in treating nonhealing diabetic foot ulcers over a 5-year period. MAIN RESULTS: The average 5-year direct wound care cost per modality and QALYs were PRP gel, $15,159 (2.87); saline gel, $33,214 (2.70); standard of care, $40,073 (2.65); noncontact kilohertz ultrasound therapy, $32,659 (2.73); human fibroblast-derived dermal substitute, $40,569 (2.65); allogenic bilayered culture skin substitute, $24,374 (2.79); bilayered cellular matrix, $37,340 (2.71); negative pressure wound therapy, $20,964 (2.81); and recombinant human platelet-derived growth factor BB, $47,252 (2.69). CONCLUSION: Use of PRP gel resulted in improved quality of life and lower cost of care over a 5-year period than other treatment modalities for nonhealing diabetic foot ulcers. Although actual treatment outcomes may differ from those modeled, PRP gel represents a potentially attractive treatment alternative for insurers and health care providers to address the cost burden and health effects of nonhealing diabetic foot ulcers.

Spironolactone-induced degradation of the TFIIH core complex XPB subunit suppresses NF-κB and AP-1 signalling.

Aims: Spironolactone (SPL) improves endothelial dysfunction and survival in heart failure. Immune modulation, including poorly understood mineralocorticoid receptor (MR)-independent effects of SPL might contribute to these benefits and possibly be useful in other inflammatory cardiovascular diseases such as pulmonary arterial hypertension. Methods and results: Using human embryonic kidney cells (HEK 293) expressing specific nuclear receptors, SPL suppressed NF-κB and AP-1 reporter activity independent of MR and other recognized nuclear receptor partners. NF-κB and AP-1 DNA binding were not affected by SPL and protein synthesis blockade did not interfere with SPL-induced suppression of inflammatory signalling. In contrast, proteasome blockade to inhibit degradation of xeroderma pigmentosum group B complementing protein (XPB), a subunit of the general transcription factor TFIIH, or XPB overexpression both prevented SPL-mediated suppression of inflammation. Similar to HEK 293 cells, a proteasome inhibitor blocked XPB loss and SPL suppression of AP-1 induced target genes in human pulmonary artery endothelial cells (PAECs). Unlike SPL, eplerenone (EPL) did not cause XPB degradation and failed to similarly suppress inflammatory signalling. SPL combined with siRNA XPB knockdown further reduced XPB protein levels and had the greatest effect on PAEC inflammatory gene transcription. Using chromatin-immunoprecipitation, PAEC target gene susceptibility to SPL was associated with low basal RNA polymerase II (RNAPII) occupancy and TNFα-induced RNAPII and XPB recruitment. XP patient-derived fibroblasts carrying an N-terminal but not C-terminal XPB mutations were insensitive to both SPL-mediated XPB degradation and TNFα-induced target gene suppression. Importantly, SPL treatment decreased whole lung XPB protein levels in a monocrotaline rat model of pulmonary hypertension and reduced inflammatory markers in an observational cohort of PAH patients. Conclusion: SPL has important anti-inflammatory effects independent of aldosterone and MR, not shared with EPL. Drug-induced, proteasome-dependent XPB degradation may be a useful therapeutic approach in cardiovascular diseases driven by inflammation.

Role of the carboxy-terminal transactivation domain and active transcription in the ligand-induced and ligand-independent degradation of the mouse Ahb-1 receptor.

To assess the importance of transactivation domains (TAD), DNA binding and transcription on the degradation of the AH receptor (AHR), Hepa-1 cells were pre-treated with actinomycin D (AD) or cycloheximide (CHX) and exposed to 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD). AD or CHX did not affect nuclear localization or DNA binding of the AHR but inhibited ligand-induced degradation. In contrast, AD or CHX did not inhibit geldanamycin (GA) induced degradation of the AHR. To assess the role of the COOH-terminal TAD in AHR degradation, stop codons were placed at nucleotide 1501 and 1921 of the Ah(b-1) AHR coding region to generate AHR(500) and AHR(640). Stable cell lines were generated and exposed to TCDD. Cells expressing AHR(500) did not induce CYP1A1 protein, but exhibited significant degradation of AHR(500). Cells expressing AHR(640) induced CYP1A1 protein to 50% of the level of cells expressing wild type AHR and exhibited significant degradation of AHR(640). Importantly, AD and CHX did not inhibit the TCDD-induced degradation of either AHR(500) and AHR(640) and these receptors showed a more rapid profile of ligand-induced degradation compared to cells expressing wild type AHR. TCDD exposure to Hepa-1 cells with reduced aryl hydrocarbon receptor nuclear translocator (ARNT), showed ligand-induced degradation of the AHR that was not blocked by AD. However, AD inhibited TCDD-induced degradation when ARNT expression was restored. These results show that multiple mechanisms exist for the ligand and GA-induced degradation of the AHR and suggest that ligand-induced degradation can switch between two mechanisms depending on the presence of a functional TAD and the binding to DNA.

Circadian regulation of a viral gene promoter in live transgenic mice expressing firefly luciferase.

PURPOSE: This study was conducted to test for possible circadian control of viral infection in live animals using bioluminescence imaging of a firefly luciferase transgene. METHODS: Transgenic mice expressing the firefly luciferase gene under the control of the promoter and enhancer of the human cytomegalovirus major immediate-early gene (CMV::luc) were examined through whole-animal imaging. Mice were crossed with HRS/J hairless albino mice to improve imaging of deep structures. RESULTS: Transgene expression in the extremities and head was elevated around dusk in mice maintained in cycles of light and dark. Signal was also elevated during the animal's night in mice maintained in extended darkness. The viral promoter was induced during the active phase of the circadian locomotor rhythm in several tissues. Both the acinar cells and islets expressed the transgene in dissociated pancreas cultures. CONCLUSIONS: These results suggest that viruses may exploit the circadian system for optimal timing of infection at particular phases in several tissue types.

Influence of a combination of two tetrachlorobiphenyl congeners (PCB 47; PCB 77) on thyroid status, choline acetyltransferase (ChAT) activity, and short- and long-term memory in 30-day-old Sprague-Dawley rats.

The important role of thyroid hormones in growth and development, maintenance of body temperature, digestion, cardiac function, and normal brain development can be disrupted by environmental contaminants like polychlorinated biphenyls (PCB). Polychlorinated biphenyls are environmental contaminants that are widespread, persistent, lipophilic, and bioaccumulate through food webs, concentrating in adipose tissue. Placental and lactational PCB exposure of offspring causes metabolic and endocrine disruptions including hypothyroxinemia, spatial learning and memory deficits, neurochemical and neurobehavioral alterations, and reproductive problems. Previous studies in our lab using the individual congeners PCB 47 (2,2',4,4'-tetrachlorobiphenyl, ortho-substituted) and PCB 77 (3,3',4,4'-tetrachlorobiphenyl, non-ortho-substituted) have demonstrated alterations in thyroid hormone levels, alterations in brain choline acetyltransferase (ChAT) activity, and spatial learning deficits. In the present study, pregnant Sprague-Dawley rats were fed a diet with or without a mixture of PCB 47/77 at 1.25 ppm, 12.5 ppm or 25.0 ppm (w/w). Rat pups were swum in the Morris water maze four times a day on days 21-29 in order for the animals to learn the position of a submerged fixed platform. A probe test was run on day 24 (30 min after last swim) for short-term memory, and on day 29 (24 h after the last swim) for long-term memory after removal of the platform. Time spent in the quadrant previously containing the platform was recorded. Rats were decapitated on day 30, serum collected and frozen at -20 degrees. ChAT activity was measured radiometrically in basal forebrain and hippocampus. All PCB-treated animals experienced a depression in both triiodothyronine (T3) and thyroxine (T4). The present study found that all doses of PCB depressed ChAT activity in hippocampus with no significant alteration in the basal forebrain. In PCB-treated animals, short-term memory showed a trend toward improvement and long-term memory toward depression, but these trends were not significant. The consequences likely stem from endocrine disruption, especially with regard to the thyroid.

Research resource: modulators of glucocorticoid receptor activity identified by a new high-throughput screening assay.

Glucocorticoid steroids affect almost every type of tissue and thus are widely used to treat a variety of human pathological conditions. However, the severity of numerous side effects limits the frequency and duration of glucocorticoid treatments. Of the numerous approaches to control off-target responses to glucocorticoids, small molecules and pharmaceuticals offer several advantages. Here we describe a new, extended high-throughput screen in intact cells to identify small molecule modulators of dexamethasone-induced glucocorticoid receptor (GR) transcriptional activity. The novelty of this assay is that it monitors changes in both GR maximal activity (A(max)) and EC(50) (the position of the dexamethasone dose-response curve). Upon screening 1280 chemicals, 10 with the greatest changes in the absolute value of A(max) or EC(50) were selected for further examination. Qualitatively identical behaviors for 60% to 90% of the chemicals were observed in a completely different system, suggesting that other systems will be similarly affected by these chemicals. Additional analysis of the 10 chemicals in a recently described competition assay determined their kinetically defined mechanism and site of action. Some chemicals had similar mechanisms of action despite divergent effects on the level of the GR-induced product. These combined assays offer a straightforward method of identifying numerous new pharmaceuticals that can alter GR transactivation in ways that could be clinically useful.

Deducing the temporal order of cofactor function in ligand-regulated gene transcription: theory and experimental verification.

Cofactors are intimately involved in steroid-regulated gene expression. Two critical questions are (1) the steps at which cofactors exert their biological activities and (2) the nature of that activity. Here we show that a new mathematical theory of steroid hormone action can be used to deduce the kinetic properties and reaction sequence position for the functioning of any two cofactors relative to a concentration limiting step (CLS) and to each other. The predictions of the theory, which can be applied using graphical methods similar to those of enzyme kinetics, are validated by obtaining internally consistent data for pair-wise analyses of three cofactors (TIF2, sSMRT, and NCoR) in U2OS cells. The analysis of TIF2 and sSMRT actions on GR-induction of an endogenous gene gave results identical to those with an exogenous reporter. Thus new tools to determine previously unobtainable information about the nature and position of cofactor action in any process displaying first-order Hill plot kinetics are now available.

Inferring mechanisms from dose-response curves.

The steady state dose-response curve of ligand-mediated gene induction usually appears to precisely follow a first-order Hill equation (Hill coefficient equal to 1). Additionally, various cofactors/reagents can affect both the potency and the maximum activity of gene induction in a gene-specific manner. Recently, we have developed a general theory for which an unspecified sequence of steps or reactions yields a first-order Hill dose-response curve (FHDC) for plots of the final product versus initial agonist concentration. The theory requires only that individual reactions "dissociate" from the downstream reactions leading to the final product, which implies that intermediate complexes are weakly bound or exist only transiently. We show how the theory can be utilized to make predictions of previously unidentified mechanisms and the site of action of cofactors/reagents. The theory is general and can be applied to any biochemical reaction that has a FHDC.

Analysis of Ah receptor-ARNT and Ah receptor-ARNT2 complexes in vitro and in cell culture.

ARNT and ARNT2 proteins are expressed in mammalian and aquatic species and exhibit a high level of amino acid identity in the basic-helix loop-helix PER/ARNT/SIM domains involved in protein interactions and DNA binding. Since the analysis of ARNT2 function at the protein level has been limited, ARNT2 function in aryl hydrocarbon receptor (AHR)-mediated signaling was evaluated and compared to ARNT. In vitro, ARNT and ARNT2 dimerized equally with the AHR in the presence of 2,3,7,8-tetracholorodibenzo-p-dioxin (TCDD) and ARNT2 outcompeted ARNT for binding to the AHR when expressed in excess. In contrast, activation of the AHR with 3-methylcholanthrene or benzo[a]pyrene resulted in predominant formation of AHR*ARNT complexes. ARNT2 expressed in Hepa-1 cell culture lines with reduced ARNT protein resulted in minimal induction of endogenous CYP1A1 protein compared to cells expressing ARNT, and mutation of the putative proline residue at amino acid 352 to histidine failed to produce an ARNT2 that could function in AHR-mediated signaling. However, the expression of ARNT2 in wild-type Hepa-1 cells reduced TCDD-mediated induction of endogenous CYP1A1 protein by 30%, even though AHR*ARNT2 complexes could not be detected in nuclear extracts. Western blot analysis of numerous mouse tissues and various cell culture lines showed that both endogenous ARNT and ARNT2 could be detected in cells derived from kidney, central nervous system, and retinal epithelium. Thus, ARNT2 has the ability to dimerize with the liganded AHR in vitro and is influenced by the activating ligand yet appears to be limited in its ability to influence AHR-mediated signaling in cell culture.