New approaches to treat ovarian cancer, the fifth leading cause of cancer mortality among women, are being sought, with the targeting of epigenetic modulators now receiving much attention. The histone acetyltransferase HBO1 functions in regulating diverse molecular processes, including DNA repair, transcription and replication, and is highly expressed in primary ovarian cancer. Here we define both the molecular function and a role in cell biomechanics for HBO1 in ovarian cancer. HBO1 preferentially acetylates histone H4 through the concomitant overexpression of co-regulator JADE2, and is required for the expression of YAP1, an ovarian cancer oncogene and mechano-transductor signaling factor. HBO1 appears therefore to have a role in determining the mechano-phenotype in ovarian cancer cells, through both signal transduction processes, and the modulation of cell elasticity as observed using direct measurements on live cells via atomic force microscopy.

IMPORTANCE Amyotrophic lateral sclerosis (ALS) is a common adult-onset neurodegenerative disease characterized by selective loss of upper and lower motor neurons. Patients with ALS have persistent peripheral and central inflammatory responses including abnormally functioning T cells and activated microglia. However, much less is known about the inflammatory gene profile of circulating innate immune monocytes in these patients.OBJECTIVE To characterize the transcriptomics of peripheral monocytes in patients with ALS.DESIGN, SETTING, AND PARTICIPANTS Monocytes were isolated from peripheral blood of 43 patients with ALS and 22 healthy control individuals. Total RNA was extracted from the monocytes and subjected to deep RNA sequencing, and these results were validated by quantitative reverse transcription polymerase chain reaction.MAIN OUTCOMES AND MEASURES The differential expressed gene signatures of these monocytes were identified using unbiased RNA sequencing strategy for gene expression profiling.RESULTS The demographics between the patients with ALS (mean [SD] age, 58.8 [1.57] years; 55.8% weremen and 44.2% were women; 90.7% were white, 4.65% were Hispanic, 2.33% were black, and 2.33% were Asian) and control individuals were similar (mean [SD] age, 57.6 [2.15] years; 50.0% were men and 50.0% were women; 90.9% were white, none were Hispanic, none were black, and 9.09% were Asian). RNA sequencing data from negative selected monocytes revealed 233 differential expressed genes in ALS monocytes compared with healthy control monocytes. Notably, ALS monocytes demonstrated a unique inflammation-related gene expression profile, the most prominent of which, including IL1B, IL8, FOSB, CXCL1, and CXCL2, were confirmed by quantitative reverse transcription polymerase chain reaction (IL8, mean [SE], 1.00 [0.18]; P=.002; FOSB, 1.00 [0.21]; P=.009; CXCL1, 1.00 [0.14]; P=.002; and CXCL2, 1.00 [0.11]; P=.01). Amyotrophic lateral sclerosis monocytes from rapidly progressing patients had more proinflammatory DEGs than monocytes from slowly progressing patients.CONCLUSIONS AND RELEVANCE Our data indicate that ALS monocytes are skewed toward a proinflammatory state in the peripheral circulation and may play a role in ALS disease progression, especially in rapidly progressing patients. This increased inflammatory response of peripheral immune cells may provide a potential target for disease-modifying therapy in patients with ALS.

Thyroid hormone (TH) receptors (TRs α and β) are homologous ligand-dependent transcription factors (TFs). While the TRs display distinct actions in development, metabolic regulation and other processes, comparisons of TRα and TRβ dependent gene regulation mostly reveal similar mechanisms of action and few TR subtype specific genes. Here, we show that TRα predominates in multipotent human adipose derived stem cells (hADSC) whereas TRβ is expressed at lower levels and is upregulated during hADSC differentiation. The TRs display several unusual properties in parental hADSC. First, TRs display predominantly cytoplasmic intracellular distribution and major TRα variants TRα1 and TRα2 colocalize with mitochondria. Second, knockdown experiments reveal that endogenous TRs influence hADSC cell morphology and expression of hundreds of genes in the absence of hormone, but do not respond to exogenous TH. Third, TRα and TRβ affect hADSC in completely distinct ways; TRα regulates cell cycle associated processes while TRβ may repress aspects of differentiation. TRα splice variant specific knockdown reveals that TRα1 and TRα2 both contribute to TRα-dependent gene expression in a gene specific manner. We propose that TRs work in a non-canonical and hormone independent manner in hADSC and that prominent subtype-specific activities emerge in the context of these unusual actions.

Aim: Thyroid hormones regulate metabolic response. While triiodothyronine (T3) is usually considered to be the active form of thyroid hormone, one form of diiodothyronine (3,5-T2) exerts T3-like effects on energy consumption and lipid metabolism. 3,5-T2 also improves glucose tolerance in rats and 3,5-T2 levels correlate with fasting glucose in humans. Presently, however, little is known about mechanisms of 3,5-T2 effects on glucose metabolism. Here, we set out to compare effects of T3, 3,5-T2 and another form of T2 (3,3-T2) in a mouse model of diet induced obesity and determined effects of T3 and 3,5-T2 on markers of classical insulin sensitization to understand how diiodothyronines influence blood glucose. Methods: Cell and protein based assays of thyroid hormone action. Assays of metabolic parameters in mice. Analysis of transcript and protein levels in different tissues by qRT-PCR and western blot. Results: T3 and 3,5-T2 both reduce body weight, adiposity and body temperature despite increased food intake. 3,3’-T2 lacks these effects. T3 and 3,5-T2 reduce blood glucose levels whereas 3,3’-T2 worsens glucose tolerance. Neither T3 or 3,5-T2 affects markers of insulin sensitization in skeletal muscle or white adipose tissue (WAT) but both reduce hepatic GLUT2 glucose transporter levels and glucose output. T3 and 3,5-T2 also induce expression of mitochondrial uncoupling proteins (UCPs) 3 and 1 in skeletal muscle and WAT, respectively. A cc ep te d A rt ic le This article is protected by copyright. All rights reserved. Conclusions: 3,5-T2 influences glucose metabolism in a manner that is distinct from insulin sensitization and involves reductions in hepatic glucose output and changes in energy utilization.

Biological processes require close cooperation of multiple transcription factors that integrate different signals. Thyroid hormone receptors (TRs) induce Krüppel-like factor 9 (KLF9) to regulate neurogenesis. Here, we show that triiodothyronine (T3) also works through TR to induce KLF9 in HepG2 liver cells, mouse liver, and mouse and human primary hepatocytes and sought to understand TR/KLF9 network function in the hepatocyte lineage and stem cells. Knockdown experiments reveal that KLF9 regulates hundreds of HepG2 target genes and modulates T3 response. Together, T3 and KLF9 target genes influence pathways implicated in stem cell self-renewal and differentiation, including Notch signaling, and we verify that T3 and KLF9 cooperate to regulate key Notch pathway genes and work independently to regulate others. T3 also induces KLF9 in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSC) and this effect persists during differentiation to definitive endoderm and hiPSC-derived hepatocytes. Microarray analysis reveals that T3 regulates hundreds of hESC and hiPSC target genes that cluster into many of the same pathways implicated in TR and KLF9 regulation in HepG2 cells. KLF9 knockdown confirms that TR and KLF9 cooperate to regulate Notch pathway genes in hESC and hiPSC, albeit in a partly cell-specific manner. Broader analysis of T3 responsive hESC/hiPSC genes suggests that TRs regulate multiple early steps in ESC differentiation. We propose that TRs cooperate with KLF9 to regulate hepatocyte proliferation and differentiation and early stages of organogenesis and that TRs exert widespread and important influences on ESC biology.

Thiazolidinediones (TZDs) are peroxisome proliferator-activated receptor gamma (PPARγ) agonists that improve insulin resistance but trigger side effects such as weight gain, edema, congestive heart failure and bone loss. GQ-16 is a PPARγ partial agonist that improves glucose tolerance and insulin sensitivity in mouse models of obesity and diabetes without inducing weight gain or edema. It is not clear whether GQ-16 acts as a partial agonist at all PPARγ target genes, or whether it displays gene-selective actions. To determine how GQ-16 influences PPARγ activity on a gene by gene basis, we compared effects of rosiglitazone (Rosi) and GQ-16 in mature 3T3-L1 adipocytes using microarray and qRT-PCR. Rosi changed expression of 1156 genes in 3T3-L1, but GQ-16 only changed 89 genes. GQ-16 generally showed weak effects upon Rosi induced genes, consistent with partial agonist actions, but a subset of modestly Rosi induced and strongly repressed genes displayed disproportionately strong GQ-16 responses. PPARγ partial agonists MLR24 and SR1664 also exhibit disproportionately strong effects on transcriptional repression. We conclude that GQ-16 displays a continuum of weak partial agonist effects but efficiently represses some negatively regulated PPARγ responsive genes. Strong repressive effects could contribute to physiologic actions of GQ-16.

Non-steroidal anti-inflammatory drugs (NSAIDs) display anti-inflammatory, antipyretic and analgesic properties by inhibiting cyclooxygenases and blocking prostaglandin production. Previous studies, however, suggested that some NSAIDs also modulate peroxisome proliferator activated receptors (PPARs), raising the possibility that such off target effects contribute to the spectrum of clinically relevant NSAID actions. In this study, we set out to understand how peroxisome proliferator activated receptor-γ (PPARγ/PPARG) interacts with NSAIDs using X-ray crystallography and to relate ligand binding modes to effects on receptor activity. We find that several NSAIDs (sulindac sulfide, diclofenac, indomethacin and ibuprofen) bind PPARγ and modulate PPARγ activity at pharmacologically relevant concentrations. Diclofenac acts as a partial agonist and binds to the PPARγ ligand binding pocket (LBP) in typical partial agonist mode, near the β-sheets and helix 3. By contrast, two copies of indomethacin and sulindac sulfide bind the LBP and, in aggregate, these ligands engage in LBP contacts that resemble agonists. Accordingly, both compounds, and ibuprofen, act as strong partial agonists. Assessment of NSAID activities in PPARγ-dependent 3T3-L1 cells reveals that NSAIDs display adipogenic activities and exclusively regulate PPARγ-dependent target genes in a manner that is consistent with their observed binding modes. Further, PPARγ knockdown eliminates indomethacin activities at selected endogenous genes, confirming receptor-dependence of observed effects. We propose that it is important to consider how individual NSAIDs interact with PPARγ to understand their activities, and that it will be interesting to determine whether high dose NSAID therapies result in PPAR activation.

The androgen receptor (AR) surface-directed antagonist MJC13 inhibits AR function and proliferation of prostate cancer (PC) cells. These effects are related to arrest of an AR/chaperone complex in the cytoplasm. Here, we compared MJC13 and classic AR antagonists such as flutamide and bicalutamide. Microarray analysis and confirmatory qRT-PCR reveals that MJC13 and flutamide inhibit dihydrotestosterone (DHT)-dependent genes in LNCaP PC cells. Both compounds are equally effective on a genome wide basis and as effective as second generation AR antagonists (MDV3100, ARN-509) at selected genes. MJC13 inhibits AR binding to the prostate specific antigen (PSA) promoter more strongly than flutamide, consistent with different mechanisms of action. Examination of efficacy of MJC13 in conditions that reflect aspects castrate resistant prostate cancer (CRPC) reveals that it inhibits flutamide activation of an AR mutant (ART877A) that emerges during flutamide withdrawal syndrome, but displays greatly restricted gene-specific activity in 22Rv1 cells that express a constitutively active truncated AR and is inactive against glucocorticoid receptor (GR), which can co-opt androgen-dependent signaling networks in CRPC. Importantly, MJC13 inhibits AR interactions with SRC2 and β-catenin in the nucleus and, unlike flutamide, strongly inhibits amplification of AR activity obtained with transfected SRC2 and β-catenin. MJC13 also inhibits DHT and β-catenin-enhanced cell division in LNCaP cells. Thus, a surface-directed antagonist can block AR activity in some conditions in which a classic antagonist fails and may display utility in particular forms of CRPC.

Abstract Background: The androgen receptor (AR) surface-directed antagonist MJC13 inhibits AR function and proliferation of prostate cancer (PC) cells and these effects are related to arrest of an AR/chaperone complex in the cytoplasm. Here, we compared activities of MJC13 and the classic AR antagonist flutamide. Method and Results: MJC13 and flutamide inhibit all dihydrotestosterone (DHT)-dependent genes in LNCaP PC cells and both compounds are equally effective. Control ChIP assays confirm that MJC13 inhibits AR binding to the prostate specific antigen (PSA) promoter more strongly than flutamide, consistent with different mechanisms of action. Examination of efficacy of MJC13 in conditions that reflect aspects of castrate resistant prostate cancer (CRPC) reveals that it inhibits a flutamide-dependent AR mutant (ART877A), but displays greatly restricted gene-specific activity in 22Rv1 cells that express a constitutively active truncated AR and is inactive against glucocorticoid receptor (GR), which can co-opt androgen-dependent signaling networks in CRPC. While flutamide inhibits AR interactions with the coactivator SRC2, it does not affect β-catenin binding. In contrast, MJC13 inhibits AR interactions with both coregulators and, unlike flutamide, strongly inhibits amplification of AR activity obtained with SRC2 and β-catenin. MJC13 also preferentially inhibits β-catenin-enhanced cell division in LNCaP cells relative to flutamide. Conclusion: Our findings suggest that a surface-directed antagonist can block AR activity in some conditions in which a classic antagonist fails and may display utility in some forms of CRPC. Citation Format: Ji Ho Suh, Arundhati Chattopadhyay, Douglas H. Sieglaff, Cheryl L. Storer, Marc B. Cox, Paul Webb. Comparison of surface-directed and classic androgen receptor antagonists. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 83. doi:10.1158/1538-7445.AM2015-83

Thyroid hormone (TH) acts through specific receptors (TRs), which are conditional transcription factors, to induce fibroblast growth factor 21 (FGF21), a peptide hormone that is usually induced by fasting and that influences lipid and carbohydrate metabolism via local hepatic and systemic endocrine effects. While TH and FGF21 display overlapping actions when administered, including reductions in serum lipids, according to the current models these hormones act independently in vivo. In this study, we examined mechanisms of regulation of FGF21 expression by TH and tested the possibility that FGF21 is required for induction of hepatic TH-responsive genes. We confirm that active TH (triiodothyronine (T3)) and the TRβ-selective thyromimetic GC1 increase FGF21 transcript and peptide levels in mouse liver and that this effect requires TRβ. T3 also induces FGF21 in cultured hepatocytes and this effect involves direct actions of TRβ1, which binds a TRE within intron 2 of FGF21. Gene expression profiles of WT and Fgf21-knockout mice are very similar, indicating that FGF21 is dispensable for the majority of hepatic T3 gene responses. A small subset of genes displays diminished T3 response in the absence of FGF21. However, most of these are not obviously directly involved in T3-dependent hepatic metabolic processes. Consistent with these results, T3-dependent effects on serum cholesterol are maintained in the Fgf21(-/-) background and we observe no effect of the Fgf21-knockout background on serum triglycerides and glucose. Our findings indicate that T3 regulates the genes involved in classical hepatic metabolic responses independently of FGF21.

There are two homologous thyroid hormone (TH) receptors (TRs α and β), which are members of the nuclear hormone receptor (NR) family. While TRs regulate different processes in vivo and other highly related NRs regulate distinct gene sets, initial studies of TR action revealed near complete overlaps in their actions at the level of individual genes. Here, we assessed the extent that TRα and TRβ differ in target gene regulation by comparing effects of equal levels of stably expressed exogenous TRs +/- T(3) in two cell backgrounds (HepG2 and HeLa). We find that hundreds of genes respond to T(3) or to unliganded TRs in both cell types, but were not able to detect verifiable examples of completely TR subtype-specific gene regulation. TR actions are, however, far from identical and we detect TR subtype-specific effects on global T(3) response kinetics in HepG2 cells and many examples of TR subtype specificity at the level of individual genes, including effects on magnitude of response to TR +/- T(3), TR regulation patterns and T(3) dose response. Cycloheximide (CHX) treatment confirms that at least some differential effects involve verifiable direct TR target genes. TR subtype/gene-specific effects emerge in the context of widespread variation in target gene response and we suggest that gene-selective effects on mechanism of TR action highlight differences in TR subtype function that emerge in the environment of specific genes. We propose that differential TR actions could influence physiologic and pharmacologic responses to THs and selective TR modulators (STRMs).

Sirtuin 1 (SIRT1) NAD+-dependent deacetylase regulates energy metabolism by modulating expression of genes involved in gluconeogenesis and other liver fasting responses. While many effects of SIRT1 on gene expression are mediated by deacetylation and activation of peroxisome proliferator activated receptor coactivator α (PGC-1α), SIRT1 also binds directly to DNA bound transcription factors, including nuclear receptors (NRs), to modulate their activity. Since thyroid hormone receptor β1 (TRβ1) regulates several SIRT1 target genes in liver and interacts with PGC-1α, we hypothesized that SIRT1 may influence TRβ1. Here, we confirm that SIRT1 cooperates with PGC-1α to enhance response to triiodothyronine, T3. We also find, however, that SIRT1 stimulates TRβ1 activity in a manner that is independent of PGC-1α but requires SIRT1 deacetylase activity. SIRT1 interacts with TRβ1 in vitro, promotes TRβ1 deacetylation in the presence of T3 and enhances ubiquitin-dependent TRβ1 turnover; a common response of NRs to activating ligands. More surprisingly, SIRT1 knockdown only strongly inhibits T3 response of a subset of TRβ1 target genes, including glucose 6 phosphatase (G-6-Pc), and this is associated with blockade of TRβ1 binding to the G-6-Pc promoter. Drugs that target the SIRT1 pathway, resveratrol and nicotinamide, modulate T3 response at dual TRβ1/SIRT1 target genes. We propose that SIRT1 is a gene-specific TRβ1 co-regulator and TRβ1/SIRT1 interactions could play important roles in regulation of liver metabolic response. Our results open possibilities for modulation of subsets of TR target genes with drugs that influence the SIRT1 pathway.

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.

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.

In anautogenous mosquitoes, egg development requires blood feeding and as a consequence mosquitoes act as vectors of numerous devastating diseases of humans and domestic animals. Understanding the molecular mechanisms regulating mosquito egg development may contribute significantly to the development of novel vector-control strategies. Previous studies have shown that in the yellow fever mosquito Aedes aegypti, nuclear receptors (NRs) play a key role in the endocrine regulation of reproduction. However, many mosquito NRs remain uncharacterized, some of which may play an important role in mosquito reproduction. Publication of the genome of A. aegypti allowed us to identify all NRs in this mosquito based on their phylogenetic relatedness to those within Insecta. We have determined that there are 20 putative A. aegypti NRs, some of which are predicted to have different isoforms. As the first step toward analysis of this gene family, we have established their expression within the two main reproductive tissues of adult female mosquitoes: fat body and ovary. All NR transcripts are present in both tissues, most displaying dynamic expression profiles during reproductive cycles. Finally, in vitro assays with isolated fat bodies were conducted to identify the role of the steroid hormone 20-hydroxyecdysone in modulating the expression of A. aegypti NRs. These data which describe the identification, expression and hormonal regulation of 20 NRs in the yellow fever mosquito lay a solid foundation for future studies on the hormonal regulation of reproduction in mosquitoes.

Ecdysteroids are multifunctional hormones in male and female arthropods and are stored in oocytes for use during embryogenesis. Ecdysteroid biosynthesis and its hormonal regulation are demonstrated for insect gonads, but not for the gonads of other arthropods. The Y-organ in the cephalothorax of crustaceans and the integument of ticks are sources of secreted ecdysteroids in adults, as in earlier stages, but the tissue source is not known for adults in many arthropod groups. Ecdysteroid metabolism occurs in several tissues of adult arthropods. This review summarizes the evidence for ecdysteroid biosynthesis by gonads and its metabolism in adult arthropods and considers the apparent uniqueness of ecdysteroid hormones in arthropods, given the predominance of vertebrate-type steroids in sister invertebrate groups and vertebrates.